From Novartis Pharma, CH-4002 Basel, Switzerland
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ABSTRACT |
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Perregaux and Gabel (Perregaux, D., and Gabel,
C. A. (1994) J. Biol. Chem. 269, 15195-15203)
reported that potassium depletion of lipopolysaccharide-stimulated
mouse macrophages induced by the potassium ionophore, nigericin, leads
to the rapid release of mature interleukin-1 (IL-1
). We have now
shown a similar phenomenon in lipopolysaccharide-stimulated human
monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa
IL-1
occurred, in the presence of nigericin (4-16
µM). No effects on the release of tumor necrosis
factor-
, IL-6, or proIL-1
were seen. Addition of the irreversible
interleukin-1
-converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1
release and activation of p45 ICE precursor. The radicicol analog itself did not inhibit ICE, but markedly, and very rapidly depleted intracellular levels of 31-kDa proIL-1
. By contrast, dexamethasone, cycloheximide, and the
Na+/H+ antiporter inhibitor,
5-(N-ethyl-N-isopropyl)amiloride, had no effect on nigericin-induced release of IL-1
. We have therefore shown
conclusively, for the first time, that nigericin-induced release of
IL-1
is dependent upon activation of p45 ICE processing. So far, the
mechanism by which reduced intracellular potassium ion concentration
triggers p45 ICE processing is not known, but further investigation in
this area could lead to the discovery of novel molecular targets
whereby control of IL-1
production might be effected.
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INTRODUCTION |
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Interleukin-1
(IL-1
)1 is produced as an
inactive 31-kDa precursor protein through the enzymatic cleavage of
IL-1
-converting enzyme (ICE), which cleaves the IL-1
precursor
between Asp-116 and Ala-117 (1). ICE itself is produced as a 45-kDa
precursor, which has recently been shown to be converted
autocatalytically to an active p10/p20 heterodimer (2). The
physiological control of ICE processing, and hence IL-1
conversion
and secretion, is still unknown. Studies by Perregaux et al.
(3, 4) suggest that IL-1
processing is controlled by intracellular
potassium ion concentration. Mouse peritoneal macrophages stimulated
with LPS produce massive amounts of cell-associated, 31-kDa IL-1
. Upon addition of the K+/H+ ionophore,
nigericin, rapid and complete processing of intracellular IL-1
occurred with the appearance of mature 17-kDa IL-1
in the medium.
Similar effects were reported using human peripheral blood monocytes.
Although in these studies marked leakage of the cytoplasmic enzyme,
lactic acid dehydrogenase (LDH) occurred, suggesting substantial cell
damage, it was argued that the effect of nigericin was not due simply
to lysis, inasmuch as, unlike the effects of hypotonic shock, at no
time were significant levels of proIL-1
detected in the culture
medium. Furthermore, the nigericin-induced 17-kDa IL-1
was shown to
have the expected N-terminal sequence. These results, together with
studies by Walev et al. (5) showing that high extracellular
concentrations of K+ or combinations of
K+-channel blockers prevented the physiological release of
IL-1
, suggest that a net reduction of intracellular K+
ion concentration is necessary for the processing of proIL-1
. Both
Perregaux et al. (4) and Walev et al. (5)
speculated that a reduction of K+ ion concentration might
activate ICE or promote the processing of pro-ICE. Alternatively, it
was suggested that nigericin-induced K+ depletion alters
the cytoplasmic compartmentalization of ICE and IL-1
. So far,
however, there has not been any direct evidence that nigericin-induced
release of IL-1
is ICE-dependent.
In the present study, we show that nigericin evokes a massive and rapid
release of 17-kDa IL-1 from prestimulated THP-1 cells under
conditions where LDH leakage is absent. Under these conditions, the
nigericin-induced secretion of IL-1
is almost completely blocked by
the irreversible ICE inhibitor,
2-valyl-alanyl-3(S)-3-amino-4-oxo-5-(2,6-dichlorobenzoyloxopentanoic) acid (Z-VAD-DCB) (6), as well as a radicicol analog (7), demonstrating
the ICE dependence of the process.
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EXPERIMENTAL PROCEDURES |
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Compounds-- Z-VAD-DCB was synthesized in our laboratories and radicicol analog A, C20H24O8 ((7S,12S,13S)-(9Z,15E)-4,12,13-trihydroxy1,2-dimethoxy-7-methyl-8,12,13,14-tetrahydro-7H-6-oxabenzocyclotetradecene-5,11-dione), was isolated from the fungus strain F/87-2509.04. The chemical structures of both compounds are shown in Fig. 1.
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Cytokine Production by THP-1 Cells and Biochemical
Assays--
Cells from the human monocytic leukemia cell line, THP-1,
were grown in RPMI medium supplemented with 110 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and
2 g/liter NaHCO3. Heat-treated fetal bovine serum (5%) was
added before use. The cells were grown to a density of 5 × 105/ml and then stimulated with interferon- (100 units/ml). Three hours later, LPS (5 µg/ml) was added. This time
point was designated time 0. Incubation continued for an additional
40 h. The media were then removed and clarified by centrifugation
at 1000 × g for 10 min. LDH measurements were
performed immediately (8). Cytokine assays were performed using
commercially available enzyme-linked immunosorbent assay kits (IL-1
,
Cayman, Ann Arbor, MI; proIL-1
, Cistron, Biotechnology, Pine Brook,
NJ; IL-6 and TNF-
, Innogenetics, Zwijndrecht, Belgium). DNA was
assayed fluorimetrically using the method of Kapuscinsiki et
al. (9).
In Vitro Refolding and Activation of ICE Processing-- Refolding and the induction of autoprocessing was carried out as described before (2) with the exception that no glutathione (GSH) was present during the dialysis step. A 3-h incubation at room temperature in the presence of 25 mM GSH following dialysis led to the induction of autocatalytic processing. Radicicol analog A was added at 5 µM final concentration to this last step. Cations, where mentioned, were present at the indicated concentrations in the refolding mixture, the dialysis buffer, as well as during the last incubation at room temperature. Western blot analysis was performed using anti-N-terminal p45 ICE or anti-p10 ICE subunit antibodies raised in our laboratories and shown to cross-react with p45 ICE. Detection was performed using an anti-rabbit IgG-POD (Sigma) with the chemiluminescence detection system of Boehringer Mannheim. Western blots were analyzed with a Molecular Dynamics Computing Densitometer 300A using Image Quant software.
IL-1 Convertase Activity Determinations--
A fluorogenic
Z-Val-Ala-Asp-aminomethyl coumarin (Z-VAD-AMC) substrate was used to
assay activity. Free AMC, which is cleaved off directly by ICE, was
detected using an excitation wavelength of 365 nm and monitoring the
emission at 450 nm. Assay conditions were as described in Refs. 1 and
6.
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RESULTS |
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Nigericin-induced IL-1 Secretion from THP-1
Cells--
Preliminary experiments showed that when THP-1 cells,
prestimulated with 5 µg/ml LPS for 39.5 h, were exposed for 30 min to nigericin, a consistent, rapid,
concentration-dependent release of IL-1
into the medium
occurred (Fig. 2A). This
increase in total cumulative IL-1
in the medium varied by 2-5-fold
in different experiments. Measurement of IL-1
levels at 30, 39.5, and again at 40 h in control cultures (no nigericin), showed that
secreted IL-1
levels were at their peak and that IL-1
release
over this time was negligible. Nigericin thus stimulated a massive and
rapid release of IL-1
over and above the normal steady-state levels. When the ICE inhibitor Z-VAD-DCB was added to the cultures at 39 h
(30 min before nigericin), it was found to substantially block the
nigericin-induced IL-1
release (Fig. 2A and Table
I). The effect of nigericin was not
caused by cytotoxicity because, as shown in Fig. 2B, even at
the highest concentration used (16 µM), there was no
increase in LDH leakage over the 30 min period of exposure. Because
longer exposure to nigericin eventually does lead to signs of
cytotoxicity, the 30-min exposure was adhered to for all experiments.
The specificity of the effect on IL-1
is further indicated in Fig.
2B, as TNF-
levels were unaltered by nigericin even at
the highest concentration. Additional studies (results not shown)
indicated that nigericin does also not affect the amount of IL-6
secreted by THP-1 cells.
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Effects of IL-1 Inhibitors on Nigericin-induced IL-1
Secretion--
In a second series of experiments we compared the
effects of Z-VAD-DCB, dexamethasone, cycloheximide and radicicol analog A, a compound previously demonstrated to reduce IL-1
production by
causing mRNA instability (7, 10), on nigericin-induced IL-1
release. Tables I and II show that,
whereas Z-VAD-DCB was able to inhibit nigericin-induced release of
IL-1
, dexamethasone or cycloheximide were without effect. The
radicicol analog also blocked the effects of nigericin. Intracellular
levels of unprocessed 31-kDa IL-1
were measured in cell lysates.
Table I shows that Z-VAD-DCB had no significant effect on intracellular
levels of proIL-1
. By contrast, both the translational inhibitor,
cycloheximide (Table II), and dexamethasone (Table I) caused a
statistically significant decrease, in intracellular proIL-1
only in
the nigericin-treated cells. A third pattern of inhibition was observed
with radicicol analog A, which markedly inhibited the levels of
proIL-1
in both control and nigericin-treated cells. Although no
increase in LDH leakage was detected, we wished to determine whether
any proIL-1
was released from the cells, which would indicate that
the nigericin-induced release of 17-kDa IL-1
was simply a result of
cellular membrane damage. The results also show that, in control cells,
only very small amounts of proIL-1
are released into the medium and
that none of the test compounds alone affected the amount released. Nigericin, however, more than doubled the concentration of proIL-1
in the medium, suggesting that a degree of non-physiological leakage of
proIL-1
did occur. The release of proIL-1
was unaffected by
either the ICE inhibitor, Z-VAD-DCB, cycloheximide or dexamethasone; however, radicicol analog A caused a marked reduction in release.
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Activation of p45 ICE Processing by Nigericin--
Because the
release of mature 17-kDa IL-1 appeared to be dependent on ICE
activity, we next investigated whether there was evidence that p45 ICE
precursor was being activated by nigericin. THP-1 cells were treated
with a combination of 100 units/ml interferon-
and 5 µg/ml LPS as
described under "Experimental Procedures." Z-VAD-DCB (1 µM) or radicicol analog A (1 µM) were given
30 min prior to nigericin (39 h after LPS addition). Following the
addition of 16 µM nigericin (39.5 h after LPS addition)
for the final 30 min of incubation, a marked and statistically
significant decrease in the amount of p45 ICE as determined by Western
blotting was observed, suggesting that processing of p45 ICE had indeed
been induced (Fig. 3, A and
B). This correlated well with the stimulation of IL-1
release by 16 µM nigericin in these experiments (Fig. 3C). In the presence of the ICE inhibitor, which we had
shown previously to inhibit autocatalytic processing of p45 ICE in a cell-free system (2), the effect of nigericin was reversed. The same
was the case for radicicol analog A. Radicicol analog A can also affect
the autocatalytic processing of p45 ICE. As Fig.
4A shows, radicicol analog A
(5 µM) when given at the same time as glutathione (GSH,
25 mM), which induces autocatalysis (2), prevents
autoprocessing and as expected, leads to the absence of ICE activity
(Fig. 4B). Unlike Z-VAD-DCB, however, radicicol analog A
does not inhibit active, processed ICE in an isolated enzyme assay
(results not shown).
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Effects of Cations on ICE Activation and Activity-- Because the main effect of nigericin is to decrease the intracellular concentration of K+ ions, it was possible that ICE activity or ICE processing could be directly affected by K+. We therefore measured recombinant ICE activity in the presence of a range of K+ ion concentrations from zero to approximately 2-fold intracellular. It was found that 200 mM K+ causes 50% inhibition of ICE activity (results not shown). However, this was not ion-specific, and similar effects were seen with Na+ and Ca2+ at comparable ionic strengths. We next looked at the effect of cations on ICE processing. Using recombinant p45 ICE under reducing conditions where autocatalytic processing is known to take place (see Ref. 2 and "Experimental Procedures"), the appearance of the p10 subunit and the resulting ICE activity in the presence of various cations was measured. Again an inhibitory effect was observed (Table III). This result clearly demonstrates that K+ ions do inhibit p45 ICE autoprocessing. In contrast to cellular systems, where monensine, A23187 (3), and amiloride (see below) have no effect, inhibition of autoprocessing in vitro is not ion-specific.
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Effects of Amiloride on Nigericin-induced IL-1
Release--
Based on findings that 5-dimethyl amiloride was able to
suppress IL-1
secretion from LPS-activated monocytes with an
IC50 = 3.5 µM, it was reported that
extracellular Na+ and high intracellular pH was required
for IL-1
secretion (12). It was possible, therefore, that the
effects of nigericin were caused by a secondary compensatory influx of
Na+ ions. If this were indeed so, one would expect
5-dimethyl amiloride to reverse or neutralize the effects of nigericin.
We thus tested IL-1
secretion from THP-1 cells in the presence of
both nigericin and 5-dimethyl amiloride given at the same time. No
reversal of the effects of nigericin by 5-dimethyl amiloride up to 30 µM was observed, suggesting that nigericin exerts its
effects directly through K+ efflux. It may well be that the
efflux of K+ is a stronger signal for ICE activation than
extracellular Na+ levels or changes in intracellular pH. It
is also quite possible that THP-1 cells react differently to these
changes, because 5-dimethyl amiloride, when given alone at 30 µM before LPS stimulation, decreased mature IL-1
secretion only slightly, which is in contrast to the strong inhibition
reported on monocytes (12).
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DISCUSSION |
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This study expands previous findings that nigericin is able to
specifically induce the release of mature IL-1 from mononucleic cells to THP-1 cells. The process was extremely rapid, with 2-5 times
as much IL-1
being released in 30 min as the cumulative release of
IL-1
over the previous 39.5 h. Unlike the studies of Perregaux
et al. (3, 4), there was no evidence of cytotoxicity over
the time course used, as indicated by the lack of any increased LDH in
the medium. On a molar basis, over 80% of the IL-1
released by
LPS-stimulated cells is in the processed 17-kDa form. Under the
influence of nigericin, the ratio of released 17-kDa to 31-kDa IL-1
in the medium remains the same, further substantiating the conclusion
that the increase is not a result of cell lysis or simple leakage.
Furthermore, the lack of effect on other cytokines such as TNF-
and
IL-6 (latter not shown) demonstrate that the effects are specific.
When comparing intracellular levels of proIL-1 at 39 and 40 h
after LPS stimulation no measurable increase could be observed (results
not shown). Steady-state synthesis of proIL-1
at this time is
apparently occurring at a very slow rate. Intracellular levels of
proIL-1
were only affected by nigericin in the presence of
inhibitors of de novo synthesis (dexamethasone and radicicol analog A, Table I; cycloheximide, Table II). The most likely explanation for a lack of an effect with nigericin alone is the presence of a homeostatic mechanism designed to maintain constant levels of intracellular proIL-1
. This would also explain why the
presence of an ICE inhibitor does not increase intracellular proIL-1
levels. Because ICE inhibitors do not lead to a decrease in proIL-1
levels inside the cell, no signal is generated to induce an increase in
the rate of proIL-1
synthesis.
The addition of an irreversible ICE inhibitor, Z-VAD-DCB, substantially
blocks the nigericin-induced release of mature IL-1, suggesting that
nigericin is dependent upon mechanisms that operate during the
physiological release of IL-1
. Analyses of the cellular levels of
p45 ICE by Western blotting clearly show that nigericin induces the
autocatalytic processing of p45 ICE (Fig. 3). The nigericin-induced
processing of p45 ICE is prevented in the presence of the ICE
inhibitor, which is consistent with the observations that this
inhibitor prevents the autocatalysis of recombinant p45 ICE in a
cell-free system (2). Furthermore, although Z-VAD-DCB also inhibits
other caspases, ICE, with the exception of caspase 4 (which cleaves
proIL-1
250-fold less effectively), is the only caspase known to
correctly cleave proIL-1
to its mature form (13). Also, unlike other
caspases, again with the exception of caspase 4, no enzyme has so far
been described to process p45 ICE other than ICE itself whereas
in vitro, ICE can also cleave pro-caspase 4. This, together
with the inhibition of p45 ICE autoprocessing, but not p10/p20 ICE
activity by radicicol analog A (see below), further reduces the
likelihood of a nigericin-induced activation of an enzymatic cascade
upstream of ICE.
Not surprisingly, 1 nM dexamethasone (a concentration shown
to give >80% inhibition of IL-1 secretion if added before LPS stimulation) and cycloheximide do not prevent nigericin-induced IL-1
processing indicating that nigericin induced secretion of IL-1
comes
from a pre-existing pool of proIL-1
.
Radicicol analog A had a profound effect on IL-1 levels. A 1-h
exposure led to a dramatic reduction in intracellular proIL-1
. Previous studies (10) have shown that radicicol analog A induces the
rapid degradation of cytokine mRNAs (including IL-1
), which have
in common the AUUUA instability motif in the 3'-untranslated region. In
contrast to dexamethasone and cycloheximide, the effect of radicicol
analog A on released proIL-1
from nigericin-treated cells therefore
reflects a drastically reduced pool of proIL-1
in the cells that is
available for processing (Tables I and II). Because over the time
period measured in our experiments there is no detectable increase in
IL-1
secretion, or proIL-1
leakage in control cells, it is not
surprising that no effect is seen with radicicol analog alone on the
secretion of mature IL-1
or on extracellular levels of proIL-1
.
The effect of radicicol analog A on the nigericin-induced release of
mature IL-1
is twofold. First, as mentioned before, radicicol analog
A induces rapid degradation of IL-1
mRNA and possibly also
inhibits transcription and translation. Thus, in the presence of
radicicol analog A, synthesis of proIL-1
is decreased. Because the
rate of proIL-1
production is decreased and the rate of proIL-1
consumption either by export of processed IL-1
or intracellular
degradation of proIL-1
, is unaffected by radicicol analog A,
intracellular proIL-1
levels are expected to decrease whereas the
levels of mature IL-1
secreted remain at the control levels because
radicicol analog A does not inhibit pre-existing mature ICE. Therefore,
also with the addition of nigericin, extracellular proIL-1
levels do
not increase. Second, because radicicol analog A also blocks p45 ICE
processing (Fig. 4) but does not inhibit ICE activity (results not
shown), the increase in IL-1
secretion in the presence of nigericin
is a result of the presence of more active ICE, a result of increased p45 ICE processing triggered by the lowering of K+ levels
by nigericin. Radicicol analog A blocking p45 ICE processing inhibits
the effects of nigericin, whereas cycloheximide, which also inhibits
protein synthesis but does not inhibit ICE activity or ICE processing
(results not shown), does not influence the effects of nigericin.
So far, however, there are no clues as to how a reduction in
K+ ion concentration may induce p45 ICE processing.
Autoprocessing of p45 ICE, in vitro is not sensitive to
K+ alone (Table III). Ionic strength-dependent
inhibition of ICE activity in vitro was also seen (results
not shown). Mature ICE was sensitive to salt concentrations equivalent
to the intracellular concentration of potassium. Such concentrations
caused approximately 50% inhibition of ICE activity using the
synthetic substrate Z-VAD-AMC. However, this effect was not
ion-specific, so it is unlikely that this explains the specific effects
of reducing K+ ion concentration in whole cells. We have
also eliminated the possibility that nigericin has a direct effect on
ICE activity in a cell-free system (results not shown). Although it
remains unclear how nigericin induces ICE processing, this effect does not appear to require metabolically active cells because
nigericin-induced proIL-1 processing continued in azide-treated
cells (results not shown).
Whether K+ ion flux plays a role in the physiological
processing of ICE and IL-1 secretion in response to pro-inflammatory stimuli, is not clear. Walev et al. (5) showed that a
variety of manipulations that resulted in reduced intracellular levels of K+ could trigger IL-1
processing. Furthermore, high
extracellular concentrations of K+ could reverse these
effects. Combinations of tetraethylammonium and 4-aminopyridine
(potassium channel blockers) could also inhibit the physiological
release of LPS-induced IL-1
. Because no single channel blocker was
effective, even at high concentration, this suggests that multiple
channels are involved and therefore potassium channel blockers are
unlikely to be leads in the search for cytokine release inhibitors.
Taken together, our results show that nigericin-induced K+
efflux induces rapid p45 ICE processing leading to an increase in active ICE, which in turn results in a higher secretion of mature IL-1. Because nigericin treatment specifically leads to mature IL-1
release, a better understanding of the mechanism by which K+ ions control p45 ICE activation and proIL-1
processing might lead to the identification of new anti-inflammatory
drug targets.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 41-61-324-7205;
Fax: 41-61-324-4046; E-mail:
Dominique.Cheneval{at}pharma.novartis.com.
1
The following abbreviations are used: IL,
interleukin; ICE, interleukin-1-converting enzyme; LDH, lactic acid
dehydrogenase; Z-VAD-AMC, carbobenzoxy-Val-Ala-Asp-aminomethyl
coumarin; Z-VAD-DCB, 2-valyl-alanyl-3(S)-3-amino-4-oxo-5-(2,6-dichlorobenzoyloxopentanoic) acid; LPS, lipopolysaccharide; TNF-
, tumor necrosis
factor-
.
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REFERENCES |
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