Increased Mature Interleukin-1beta (IL-1beta ) Secretion from THP-1 Cells Induced by Nigericin Is a Result of Activation of p45 IL-1beta -converting Enzyme Processing*

Dominique ChenevalDagger , Paul Ramage, Tania Kastelic, Terez Szelestenyi, Heinz Niggli, René Hemmig, Martin Bachmann, and Andrew MacKenzie

From Novartis Pharma, CH-4002 Basel, Switzerland

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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-1beta (IL-1beta ). We have now shown a similar phenomenon in lipopolysaccharide-stimulated human monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa IL-1beta occurred, in the presence of nigericin (4-16 µM). No effects on the release of tumor necrosis factor-alpha , IL-6, or proIL-1beta were seen. Addition of the irreversible interleukin-1beta -converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1beta 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-1beta . 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-1beta . We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1beta 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-1beta production might be effected.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Interleukin-1beta (IL-1beta )1 is produced as an inactive 31-kDa precursor protein through the enzymatic cleavage of IL-1beta -converting enzyme (ICE), which cleaves the IL-1beta 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-1beta conversion and secretion, is still unknown. Studies by Perregaux et al. (3, 4) suggest that IL-1beta processing is controlled by intracellular potassium ion concentration. Mouse peritoneal macrophages stimulated with LPS produce massive amounts of cell-associated, 31-kDa IL-1beta . Upon addition of the K+/H+ ionophore, nigericin, rapid and complete processing of intracellular IL-1beta occurred with the appearance of mature 17-kDa IL-1beta 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-1beta detected in the culture medium. Furthermore, the nigericin-induced 17-kDa IL-1beta 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-1beta , suggest that a net reduction of intracellular K+ ion concentration is necessary for the processing of proIL-1beta . 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-1beta . So far, however, there has not been any direct evidence that nigericin-induced release of IL-1beta is ICE-dependent.

In the present study, we show that nigericin evokes a massive and rapid release of 17-kDa IL-1beta from prestimulated THP-1 cells under conditions where LDH leakage is absent. Under these conditions, the nigericin-induced secretion of IL-1beta 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 1.   Chemical structures of Z-VAD-DCB and radicicol analog A.

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-gamma (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-1beta , Cayman, Ann Arbor, MI; proIL-1beta , Cistron, Biotechnology, Pine Brook, NJ; IL-6 and TNF-alpha , 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-1beta 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Nigericin-induced IL-1beta 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-1beta into the medium occurred (Fig. 2A). This increase in total cumulative IL-1beta in the medium varied by 2-5-fold in different experiments. Measurement of IL-1beta levels at 30, 39.5, and again at 40 h in control cultures (no nigericin), showed that secreted IL-1beta levels were at their peak and that IL-1beta release over this time was negligible. Nigericin thus stimulated a massive and rapid release of IL-1beta 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-1beta 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-1beta is further indicated in Fig. 2B, as TNF-alpha 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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Fig. 2.   A, nigericin-induced IL-1beta release from THP-1 cells. Cells were stimulated as described under "Experimental Procedures." Nigericin was added 39.5 h after LPS, and measurements were made 30 min later. The concentrations of IL-1beta (normalized against cellular DNA) in the medium harvested from THP-1 cultures, 40 h after stimulation with LPS are shown. Filled squares, concentration-related increase in IL-1beta concentrations caused by treatment with nigericin over the final 30 min of culture; filled circles, ICE inhibitor, Z-VAD-DCB (1 µM) added to the cells 30 min before nigericin (p values relate to differences between nigericin ± ICE inhibitor). The result is representative of a series of experiments in which the stimulation index ± 16 µM nigericin varied from 2 to 5. In all cases, the nigericin effect was completely blocked by 1 µM Z-VAD-DCB. Given are means ± S.E., n = 4 separate cultures. B, specificity of nigericin for IL-1beta release. The effect of nigericin was specific for IL-1beta . Another inflammatory cytokine produced by THP-1 cells, TNF-alpha (filled symbols, shapes as described above) is unaffected. LDH leakage (open symbols, shapes as described above) does not increase significantly above the levels seen in the control cultures without nigericin. Given are means ± S.E., n = 4 separate cultures.

                              
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Table I
Effects of inhibitors on IL-1beta secretion and intracellular and extracellular proIL-1beta accumulation
THP-1 cells were treated as described in Fig. 2. Measurements were done by ELISA. The effect of Z-VAD-DCB (1 µM), dexamethasone (Dex, 1 nM), and radicicol analog A (1 µM) given 30 min prior to nigericin (16 µM), on nigericin-induced release of 17-kDa IL-1beta into the medium, as well as intracellular and extracellular levels of 31-kDa proIL-1beta are shown. Given are means ± S.E. (ng/µg DNA), n = 4 separate cultures. *, p < 0.05; **, p < 0.01, ***, p < 0.001. Analysis of variance was followed by Bonferoni multiple comparisons test. NS, not significant.

Effects of IL-1 Inhibitors on Nigericin-induced IL-1beta 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-1beta production by causing mRNA instability (7, 10), on nigericin-induced IL-1beta release. Tables I and II show that, whereas Z-VAD-DCB was able to inhibit nigericin-induced release of IL-1beta , dexamethasone or cycloheximide were without effect. The radicicol analog also blocked the effects of nigericin. Intracellular levels of unprocessed 31-kDa IL-1beta were measured in cell lysates. Table I shows that Z-VAD-DCB had no significant effect on intracellular levels of proIL-1beta . By contrast, both the translational inhibitor, cycloheximide (Table II), and dexamethasone (Table I) caused a statistically significant decrease, in intracellular proIL-1beta only in the nigericin-treated cells. A third pattern of inhibition was observed with radicicol analog A, which markedly inhibited the levels of proIL-1beta in both control and nigericin-treated cells. Although no increase in LDH leakage was detected, we wished to determine whether any proIL-1beta was released from the cells, which would indicate that the nigericin-induced release of 17-kDa IL-1beta was simply a result of cellular membrane damage. The results also show that, in control cells, only very small amounts of proIL-1beta 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-1beta in the medium, suggesting that a degree of non-physiological leakage of proIL-1beta did occur. The release of proIL-1beta 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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Table II
Effects of cycloheximide and nigericin on IL-1beta secretion and intracellular and extracellular proIL-1beta accumulation
THP-1 cells were treated as described in Fig. 2. Measurements were done by ELISA. The effect of cycloheximide (CHX, 20 µM) given 30 min prior to nigericin (16 µM), on nigericin-induced release of 17-kDa IL-1beta into the medium, as well as intracellular and extracellular levels of 31-kDa proIL-1beta are shown. Given are means ± S.E. (ng/µg DNA), n = 4 separate cultures. **, p < 0.01; ***, p < 0.001. Analysis of variance was followed by Bonferoni multiple comparisons test. NS, not significant.

Activation of p45 ICE Processing by Nigericin-- Because the release of mature 17-kDa IL-1beta 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-gamma 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-1beta 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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Fig. 3.   Effect of nigericin on p45 ICE activation. Western blot analysis of cellular ICE. Cells were stimulated as described under "Experimental Procedures" and harvested after 40 h of incubation in the presence of LPS. Nigericin was added 39.5 h after LPS, and Z-VAD-DCB was added 30 min prior to nigericin. Secreted mature IL-1beta was measured by enzyme-linked immunosorbent assay. To control for possible uneven blotting, equal aliquots of cells were loaded randomly onto SDS gels and levels of p45 ICE were analyzed by Western blotting using rabbit anti-human N-terminal p45 ICE antibodies that cross-react with p45 ICE. Digitized images of the autoradiographs allowed relative band intensities to be measured. Given are means ± S.E. (n = 6 for samples with nigericin, n = 12 for the control, and n = 3 for all other treatments). One-way analysis of variance statistical analysis was carried out followed by Bonferroni multiple comparison test against the control (a) and the nigericin-treated sample (b).


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Fig. 4.   Inhibition of p45 ICE processing by radicicol analog A. Recombinant p45 ICE was activated by 25 mM GSH as described under "Experimental Procedures," and the ensuing ICE activity was determined by a fluorometric assay using Z-VAD-AMC as substrate. A, lane a, autocatalytic processing of p45 ICE; lane b, autocatalytic processing of p45 ICE in the presence of 5 µM radicicol analog A. B, bar a, ICE activity of processed p 45 ICE corresponding to A, lane a; bar b, ICE activity of processed p45 ICE corresponding to A, lane b. Activity is expressed in molar concentration of AMC formed, calculated from AMC standards included in the assay.

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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Table III
Influence of cations on in vitro p45 ICE processing
Recombinant human p45 ICE was activated by 25 mM GSH as described under "Experimental Procedures" in the presence of different ions at various concentrations. The ensuing ICE activity was determined by a fluorometric assay as described under "Experimental Procedures" and is given in percent of control. Western blots were performed with a rabbit anti-human p10 ICE subunit antibody, and relative band intensities were determined by digitized imaging.

Effects of Amiloride on Nigericin-induced IL-1beta Release-- Based on findings that 5-dimethyl amiloride was able to suppress IL-1beta 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-1beta 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-1beta 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-1beta secretion only slightly, which is in contrast to the strong inhibition reported on monocytes (12).

    DISCUSSION
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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-1beta from mononucleic cells to THP-1 cells. The process was extremely rapid, with 2-5 times as much IL-1beta being released in 30 min as the cumulative release of IL-1beta 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-1beta 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-1beta 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-alpha and IL-6 (latter not shown) demonstrate that the effects are specific.

When comparing intracellular levels of proIL-1beta at 39 and 40 h after LPS stimulation no measurable increase could be observed (results not shown). Steady-state synthesis of proIL-1beta at this time is apparently occurring at a very slow rate. Intracellular levels of proIL-1beta 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-1beta . This would also explain why the presence of an ICE inhibitor does not increase intracellular proIL-1beta levels. Because ICE inhibitors do not lead to a decrease in proIL-1beta levels inside the cell, no signal is generated to induce an increase in the rate of proIL-1beta synthesis.

The addition of an irreversible ICE inhibitor, Z-VAD-DCB, substantially blocks the nigericin-induced release of mature IL-1beta , suggesting that nigericin is dependent upon mechanisms that operate during the physiological release of IL-1beta . 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-1beta 250-fold less effectively), is the only caspase known to correctly cleave proIL-1beta 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-1beta secretion if added before LPS stimulation) and cycloheximide do not prevent nigericin-induced IL-1beta processing indicating that nigericin induced secretion of IL-1beta comes from a pre-existing pool of proIL-1beta .

Radicicol analog A had a profound effect on IL-1beta levels. A 1-h exposure led to a dramatic reduction in intracellular proIL-1beta . Previous studies (10) have shown that radicicol analog A induces the rapid degradation of cytokine mRNAs (including IL-1beta ), 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-1beta from nigericin-treated cells therefore reflects a drastically reduced pool of proIL-1beta 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-1beta secretion, or proIL-1beta leakage in control cells, it is not surprising that no effect is seen with radicicol analog alone on the secretion of mature IL-1beta or on extracellular levels of proIL-1beta . The effect of radicicol analog A on the nigericin-induced release of mature IL-1beta is twofold. First, as mentioned before, radicicol analog A induces rapid degradation of IL-1beta mRNA and possibly also inhibits transcription and translation. Thus, in the presence of radicicol analog A, synthesis of proIL-1beta is decreased. Because the rate of proIL-1beta production is decreased and the rate of proIL-1beta consumption either by export of processed IL-1beta or intracellular degradation of proIL-1beta , is unaffected by radicicol analog A, intracellular proIL-1beta levels are expected to decrease whereas the levels of mature IL-1beta 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-1beta 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-1beta 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-1beta processing continued in azide-treated cells (results not shown).

Whether K+ ion flux plays a role in the physiological processing of ICE and IL-1beta 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-1beta 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-1beta . 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-1beta . Because nigericin treatment specifically leads to mature IL-1beta release, a better understanding of the mechanism by which K+ ions control p45 ICE activation and proIL-1beta processing might lead to the identification of new anti-inflammatory drug targets.

    FOOTNOTES

* 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.

Dagger 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-1beta -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-alpha , tumor necrosis factor-alpha .

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

  1. Thornberry, N. A., Bull, H. G., Calaycay, J. R., Chapman, K. T., Howard, A. D., Kostura, M. J., Miller, D. K., Molineaux, S. M., Weidner, J. R., Aunins, J., Elliston, K. O., Ayala, J. M., Casano, F. J., Chin, J., Ding, G. J.-F., Egger, L. A., Gaffney, E. P., Limjuco, G., Palyha, O. C., Raju, S. M., Rolando, A. M., Salley, J. P., Yamin, T.-T., Lee, T. D., Shively, J. E., MacCross, M., Mumford, R. A., Schmidt, J. A., and Tocci, M. J. (1992) Nature 356, 768-774[Medline] [Order article via Infotrieve]
  2. Ramage, P., Cheneval, D., Chvei, M, Graff, P., Hemmig, R., Heng, R., Kocher, H.-P, MacKenzie, A., Memmert, K., Revesz, L., and Wishart, W. (1995) J. Biol. Chem. 270, 9378-9383[Abstract/Free Full Text]
  3. Perregaux, D., Barberia, J., Lanzetti, A. J., Geoghegan, K. F., Carty, T. J., and Gabel, C. A. (1992) J. Immunol. 149, 1294-1303[Abstract/Free Full Text]
  4. Perregaux, D., and Gabel, C. A. (1994) J. Biol. Chem. 269, 15195-15203[Abstract/Free Full Text]
  5. Walev, I., Reske, K., Palmer, M., Valeva, A., and Bhakdi, S. (1995) EMBO J. 14, 1607-1614[Abstract]
  6. Loddick, S. A., MacKenzie, A., and Rothwell, N. J. (1996) Neuropharmacol. Neurotoxicol. 7, 1465-1468
  7. Kastelic, T., Schnyder, J., Leutwiler, A., Traber, R., Streit, B., Niggli, H., MacKenzie, A., and Cheneval, D. (1996) Cytokine 8, 751-761[CrossRef][Medline] [Order article via Infotrieve]
  8. Schnyder, J., Bollinger, P., and Payne, T. (1990) Agents Actions 30, 350-362[Medline] [Order article via Infotrieve]
  9. Kapuscinski, J., and Skoczylas, B. (1977) Anal. Biochem. 83, 252-257[Medline] [Order article via Infotrieve]
  10. Elford, P. R., Dixon, A. K., MacKenzie, A. R., Leutwiler, A., and Schnyder, J. (1996) Pharmacol. Commun. 7, 301-308
  11. Ayala, J. M., Yamin, T.-T., Egger, L. A., Chin, J., Kostura, M. J., and Miller, D. K. (1994) J. Immunol. 153, 2592-2599[Abstract/Free Full Text]
  12. Orlinska, U., and Newton, R. C. (1992) Am. J. Physiol. 263, C1073-C1080[Abstract/Free Full Text]
  13. Margolin, N., Raybuck, S. A., Wilson, K. P., Chen, W., Fox, T., Gu, Y., and Livingston, D. J. (1997) J. Biol. Chem. 272, 7223-7228[Abstract/Free Full Text]


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