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

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-1β (IL-1β). We have now shown a similar phenomenon in lipopolysaccharide-stimulated human monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa IL-1β occurred, in the presence of nigericin (4–16 μm). No effects on the release of tumor necrosis factor-α, IL-6, or proIL-1β were seen. Addition of the irreversible interleukin-1β-converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1β 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-1β. 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-1β. We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1β 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-1β production might be effected. 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-1β (IL-1β). We have now shown a similar phenomenon in lipopolysaccharide-stimulated human monocytic leukemia THP-1 cells. Rapid secretion of mature, 17-kDa IL-1β occurred, in the presence of nigericin (4–16 μm). No effects on the release of tumor necrosis factor-α, IL-6, or proIL-1β were seen. Addition of the irreversible interleukin-1β-converting enzyme (ICE) inhibitor, Z-Val-Ala-Asp-dichlorobenzoate, or a radicicol analog, inhibited nigericin-induced mature IL-1β 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-1β. 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-1β. We have therefore shown conclusively, for the first time, that nigericin-induced release of IL-1β 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-1β production might be effected. Interleukin-1β (IL-1β) 1The following abbreviations are used: IL, interleukin; ICE, interleukin-1β-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-α, tumor necrosis factor-α. 1The following abbreviations are used: IL, interleukin; ICE, interleukin-1β-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-α, tumor necrosis factor-α. is produced as an inactive 31-kDa precursor protein through the enzymatic cleavage of IL-1β-converting enzyme (ICE), which cleaves the IL-1β precursor between Asp-116 and Ala-117 (1Thornberry 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. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2194) Google Scholar). ICE itself is produced as a 45-kDa precursor, which has recently been shown to be converted autocatalytically to an active p10/p20 heterodimer (2Ramage P. Cheneval D. Chvei M Graff P. Hemmig R. Heng R. Kocher H.-P MacKenzie A. Memmert K. Revesz L. Wishart W. J. Biol. Chem. 1995; 270: 9378-9383Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The physiological control of ICE processing, and hence IL-1β conversion and secretion, is still unknown. Studies by Perregaux et al.(3Perregaux D. Barberia J. Lanzetti A.J. Geoghegan K.F. Carty T.J. Gabel C.A. J. Immunol. 1992; 149: 1294-1303PubMed Google Scholar, 4Perregaux D. Gabel C.A. J. Biol. Chem. 1994; 269: 15195-15203Abstract Full Text PDF PubMed Google Scholar) suggest that IL-1β processing is controlled by intracellular potassium ion concentration. Mouse peritoneal macrophages stimulated with LPS produce massive amounts of cell-associated, 31-kDa IL-1β. Upon addition of the K+/H+ ionophore, nigericin, rapid and complete processing of intracellular IL-1β occurred with the appearance of mature 17-kDa IL-1β 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-1β detected in the culture medium. Furthermore, the nigericin-induced 17-kDa IL-1β was shown to have the expected N-terminal sequence. These results, together with studies by Walev et al. (5Walev I. Reske K. Palmer M. Valeva A. Bhakdi S. EMBO J. 1995; 14: 1607-1614Crossref PubMed Scopus (234) Google Scholar) showing that high extracellular concentrations of K+ or combinations of K+-channel blockers prevented the physiological release of IL-1β, suggest that a net reduction of intracellular K+ion concentration is necessary for the processing of proIL-1β. Both Perregaux et al. (4Perregaux D. Gabel C.A. J. Biol. Chem. 1994; 269: 15195-15203Abstract Full Text PDF PubMed Google Scholar) and Walev et al. (5Walev I. Reske K. Palmer M. Valeva A. Bhakdi S. EMBO J. 1995; 14: 1607-1614Crossref PubMed Scopus (234) Google Scholar) 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-1β. So far, however, there has not been any direct evidence that nigericin-induced release of IL-1β is ICE-dependent. In the present study, we show that nigericin evokes a massive and rapid release of 17-kDa IL-1β from prestimulated THP-1 cells under conditions where LDH leakage is absent. Under these conditions, the nigericin-induced secretion of IL-1β 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) (6Loddick S.A. MacKenzie A. Rothwell N.J. Neuropharmacol. Neurotoxicol. 1996; 7: 1465-1468Google Scholar), as well as a radicicol analog (7Kastelic T. Schnyder J. Leutwiler A. Traber R. Streit B. Niggli H. MacKenzie A. Cheneval D. Cytokine. 1996; 8: 751-761Crossref PubMed Scopus (48) Google Scholar), demonstrating the ICE dependence of the process. 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. 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 mml-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-γ (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 (8Schnyder J. Bollinger P. Payne T. Agents Actions. 1990; 30: 350-362Crossref PubMed Scopus (19) Google Scholar). Cytokine assays were performed using commercially available enzyme-linked immunosorbent assay kits (IL-1β, Cayman, Ann Arbor, MI; proIL-1β, Cistron, Biotechnology, Pine Brook, NJ; IL-6 and TNF-α, Innogenetics, Zwijndrecht, Belgium). DNA was assayed fluorimetrically using the method of Kapuscinsiki et al. (9Kapuscinski J. Skoczylas B. Anal. Biochem. 1977; 83: 252-257Crossref PubMed Scopus (433) Google Scholar). Refolding and the induction of autoprocessing was carried out as described before (2Ramage P. Cheneval D. Chvei M Graff P. Hemmig R. Heng R. Kocher H.-P MacKenzie A. Memmert K. Revesz L. Wishart W. J. Biol. Chem. 1995; 270: 9378-9383Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) 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. 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. 1Thornberry 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. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2194) Google Scholar and6Loddick S.A. MacKenzie A. Rothwell N.J. Neuropharmacol. Neurotoxicol. 1996; 7: 1465-1468Google Scholar. 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-1β into the medium occurred (Fig. 2 A). This increase in total cumulative IL-1β in the medium varied by 2–5-fold in different experiments. Measurement of IL-1β levels at 30, 39.5, and again at 40 h in control cultures (no nigericin), showed that secreted IL-1β levels were at their peak and that IL-1β release over this time was negligible. Nigericin thus stimulated a massive and rapid release of IL-1β 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-1β release (Fig. 2 A and TableI). The effect of nigericin was not caused by cytotoxicity because, as shown in Fig. 2 B, 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-1β is further indicated in Fig. 2 B, as TNF-α 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.Table IEffects of inhibitors on IL-1β secretion and intracellular and extracellular proIL-1β accumulationControlZ-VAD-DCBNigericinNigericin + Z-VAD-DCBDexNigericin + DexRadicicol analog ANigericin + radicicol analog ASecreted IL-1β1.06 cells were as described in Fig. were by The effect of Z-VAD-DCB μm), and radicicol analog A 30 min to nigericin (16 μm), on nigericin-induced release of 17-kDa IL-1β into the as well as intracellular and extracellular levels of 31-kDa proIL-1β are are of was by not in a THP-1 cells were as described in Fig. were by The effect of Z-VAD-DCB μm), and radicicol analog A 30 min to nigericin (16 μm), on nigericin-induced release of 17-kDa IL-1β into the as well as intracellular and extracellular levels of 31-kDa proIL-1β are are of was by not In a of experiments we the effects of Z-VAD-DCB, dexamethasone, and radicicol analog A, a to IL-1β production by (7Kastelic T. Schnyder J. Leutwiler A. Traber R. Streit B. Niggli H. MacKenzie A. Cheneval D. Cytokine. 1996; 8: 751-761Crossref PubMed Scopus (48) Google Scholar, MacKenzie Leutwiler A. Schnyder J. 1996; 7: Scholar), on nigericin-induced IL-1β and show Z-VAD-DCB was to inhibit nigericin-induced release of IL-1β, or were The radicicol analog also blocked the effects of levels of 31-kDa IL-1β were in cell that Z-VAD-DCB had no significant effect on intracellular levels of proIL-1β. By contrast, both the inhibitor, and caused a significant in intracellular proIL-1β in the cells. A of was with radicicol analog A, which inhibited the levels of proIL-1β in both control and cells. Although no increase in LDH leakage was we to any proIL-1β was from the cells, which that the nigericin-induced release of 17-kDa IL-1β was simply a of The also show in control cells, very amounts of proIL-1β are into the medium and that of the compounds the amount however, the concentration of proIL-1β in the suggesting that a of leakage of proIL-1β did The release of proIL-1β was by the ICE inhibitor, Z-VAD-DCB, or however, radicicol analog A caused a marked reduction in of and nigericin on IL-1β secretion and intracellular and extracellular proIL-1β + cells were as described in Fig. were by The effect of 30 min to nigericin (16 μm), on nigericin-induced release of 17-kDa IL-1β into the as well as intracellular and extracellular levels of 31-kDa proIL-1β are are of was by not in a THP-1 cells were as described in Fig. were by The effect of 30 min to nigericin (16 μm), on nigericin-induced release of 17-kDa IL-1β into the as well as intracellular and extracellular levels of 31-kDa proIL-1β are are of was by not Because the release of mature 17-kDa IL-1β to be dependent on ICE we there was evidence that p45 ICE precursor was by THP-1 cells were with a of 100 units/ml interferon-γ and 5 μg/ml LPS as described under Z-VAD-DCB or radicicol analog A were 30 min to nigericin h LPS the addition of μm nigericin h LPS for the final 30 min of a marked and significant in the amount of p45 ICE as by Western was suggesting that processing of p45 ICE had been induced (Fig. A This well with the of IL-1β release by μm nigericin in these experiments (Fig. In the presence of the ICE inhibitor, which we had shown to inhibit autocatalytic processing of p45 ICE in a system (2Ramage P. Cheneval D. Chvei M Graff P. Hemmig R. Heng R. Kocher H.-P MacKenzie A. Memmert K. Revesz L. Wishart W. J. Biol. Chem. 1995; 270: 9378-9383Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), the effect of nigericin was The was the for radicicol analog A. Radicicol analog A also affect the autocatalytic processing of p45 ICE. Fig. A radicicol analog A (5 when at the time as glutathione 25 which (2Ramage P. Cheneval D. Chvei M Graff P. Hemmig R. Heng R. Kocher H.-P MacKenzie A. Memmert K. Revesz L. Wishart W. J. Biol. Chem. 1995; 270: 9378-9383Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), autoprocessing and as leads to the of ICE (Fig. Z-VAD-DCB, however, radicicol analog A does not inhibit ICE in an isolated enzyme assay (results not of p45 ICE processing by radicicol analog A. p45 ICE was by 25 as described under and the ICE was by a assay using as A, autocatalytic processing of p45 autocatalytic processing of p45 ICE in the presence of 5 μm radicicol analog A. ICE of ICE ICE of p45 ICE to A, is in concentration of from in the Image Because the effect of nigericin is to the intracellular concentration of K+ it was that ICE or ICE processing could be directly by We therefore ICE in the presence of a of K+ ion concentrations from to was found that mm of ICE (results not this was not and similar effects were with and at We at the effect of on ICE processing. p45 ICE under conditions where autocatalytic processing is to P. Cheneval D. Chvei M Graff P. Hemmig R. Heng R. Kocher H.-P MacKenzie A. Memmert K. Revesz L. Wishart W. J. Biol. Chem. 1995; 270: 9378-9383Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar and the appearance of the subunit and the ICE in the presence of was an effect was This that K+ inhibit p45 ICE In to where D. Barberia J. Lanzetti A.J. Geoghegan K.F. Carty T.J. Gabel C.A. J. Immunol. 1992; 149: 1294-1303PubMed Google Scholar), and have no of autoprocessing in not of on in p45 ICE were assayed at in ion concentrations which had no effect on ICE as in experiments (results not + human p45 ICE was by 25 mm GSH as described under in the presence of different at The ICE was by a assay as described under and is in of Western blots were performed with a ICE subunit and were by were assayed at in ion concentrations which had no effect on ICE as in experiments (results not in a human p45 ICE was by 25 mm GSH as described under in the presence of different at The ICE was by a assay as described under and is in of Western blots were performed with a ICE subunit and were by on that was to IL-1β secretion from with an it was reported that extracellular and high intracellular was for IL-1β secretion J. 1992; PubMed Google Scholar). was that the effects of nigericin were caused by a of this were to or the effects of We thus IL-1β secretion from THP-1 cells in the presence of both nigericin and at the No of the effects of nigericin by to 30 μm was suggesting that nigericin effects directly through K+ well be that the of K+ is a for ICE activation extracellular levels or in intracellular is also that THP-1 cells to these when at 30 μm before LPS mature IL-1β secretion which is in to the reported on J. 1992; PubMed Google Scholar). This that nigericin is to the release of mature IL-1β from cells to THP-1 cells. The was rapid, with as IL-1β in 30 min as the cumulative release of IL-1β over the 39.5 h. the studies of al. D. Barberia J. Lanzetti A.J. Geoghegan K.F. Carty T.J. Gabel C.A. J. Immunol. 1992; 149: 1294-1303PubMed Google Scholar, 4Perregaux D. Gabel C.A. J. Biol. Chem. 1994; 269: 15195-15203Abstract Full Text PDF PubMed Google Scholar), there was no evidence of cytotoxicity over the time as indicated by the of any LDH in the medium. a over of the IL-1β by cells is in the 17-kDa Under the of nigericin, the of 17-kDa to 31-kDa IL-1β in the medium the further the that the increase is not a of cell or Furthermore, the of effect on as TNF-α and IL-6 not shown) that the effects are When intracellular levels of proIL-1β at 39 and 40 h LPS no increase could be (results not of proIL-1β at this time is at a very levels of proIL-1β were by nigericin in the presence of inhibitors of and radicicol analog A, cycloheximide, The for a of an effect with nigericin is the presence of a mechanism to levels of intracellular proIL-1β. This also the presence of an ICE inhibitor does not increase intracellular proIL-1β levels. Because ICE inhibitors not lead to a in proIL-1β levels the no is to an increase in the of proIL-1β The addition of an irreversible ICE inhibitor, Z-VAD-DCB, substantially the nigericin-induced release of mature IL-1β, suggesting that nigericin is dependent upon that during the physiological release of IL-1β. of the levels of p45 ICE by Western show that nigericin the autocatalytic processing of p45 ICE (Fig. The nigericin-induced processing of p45 ICE is prevented in the presence of the ICE inhibitor, which is with the that this inhibitor the of p45 ICE in a system (2Ramage P. Cheneval D. Chvei M Graff P. Hemmig R. Heng R. Kocher H.-P MacKenzie A. Memmert K. Revesz L. Wishart W. J. Biol. Chem. 1995; 270: 9378-9383Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Furthermore, Z-VAD-DCB also ICE, with the exception of cleaves proIL-1β is the to proIL-1β to mature S.A. W. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). unlike again with the exception of no enzyme has been described to p45 ICE ICE itself ICE also together with the of p45 ICE but not p10/p20 ICE by radicicol analog A further the of a nigericin-induced activation of an enzymatic of ICE. nm concentration shown to of IL-1β secretion added before LPS and not nigericin-induced IL-1β processing that nigericin induced secretion of IL-1β from a of proIL-1β. Radicicol analog A had a effect on IL-1β levels. A exposure led to a reduction in intracellular proIL-1β. studies MacKenzie Leutwiler A. Schnyder J. 1996; 7: Scholar) have shown that radicicol analog A the rapid of which have in the in the In to and cycloheximide, the effect of radicicol analog A on proIL-1β from cells therefore a reduced of proIL-1β in the cells that is available for processing and Because over the time period in our experiments there is no increase in IL-1β secretion, or proIL-1β leakage in control cells, it is not that no effect is with radicicol analog on the secretion of mature IL-1β or on extracellular levels of proIL-1β. The effect of radicicol analog A on the nigericin-induced release of mature IL-1β is as radicicol analog A rapid of IL-1β and also and in the presence of radicicol analog A, of proIL-1β is Because the of proIL-1β production is and the of proIL-1β by of IL-1β or intracellular of proIL-1β, is by radicicol analog A, intracellular proIL-1β levels are expected to the levels of mature IL-1β secreted at the control levels radicicol analog A does not inhibit mature ICE. also with the addition of nigericin, extracellular proIL-1β levels not radicicol analog A also p45 ICE processing (Fig. but does not inhibit ICE (results not the increase in IL-1β secretion in the presence of nigericin is a of the presence of active ICE, a of p45 ICE processing by the of K+ levels by Radicicol analog A p45 ICE processing the effects of nigericin, cycloheximide, which also protein but does not inhibit ICE or ICE processing (results not does not the effects of So far, however, there are no as to a reduction in K+ ion concentration p45 ICE processing. of p45 ICE, in is not to K+ of ICE in was also (results not ICE was to concentrations to the intracellular concentration of concentrations caused of ICE using the substrate this effect was not it is that this the effects of K+ ion concentration in cells. We have also the that nigericin has a direct effect on ICE in a system (results not Although it nigericin ICE processing, this effect does not to active cells nigericin-induced proIL-1β processing continued in cells (results not K+ ion a in the physiological processing of ICE and IL-1β secretion in to is not Walev et al. (5Walev I. Reske K. Palmer M. Valeva A. Bhakdi S. EMBO J. 1995; 14: 1607-1614Crossref PubMed Scopus (234) Google Scholar) showed that a of that in reduced intracellular levels of K+ could IL-1β processing. Furthermore, high extracellular concentrations of K+ could these of and could also inhibit the physiological release of IL-1β. Because no was even at high this that are and therefore potassium blockers are to be leads in the for release our show that nigericin-induced rapid p45 ICE processing to an increase in active ICE, which in in a secretion of mature IL-1β. Because nigericin leads to mature IL-1β a of the mechanism by which K+ control p45 ICE activation and proIL-1β processing might lead to the of