Effects of zinc chelation on IL-1β secretion
We first tested the hypothesis that zinc contributes to caspase-1-dependent pro-IL-1β cleavage and mature IL-1β release by exposing LPS-primed (1 μg/mL, 2 h) mouse peritoneal macrophages to the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylene diamine (TPEN) for 15 min prior to 10 min incubation with 5 mM ATP. Pre-treatment of LPS-primed mouse peritoneal macrophages with TPEN abolished the release of IL-1β in response to ATP (Fig. 1Ai). This was due to inhibition of pro-IL-1β processing (Fig. 1Aii) resulting from an inhibition of caspase-1 activation, as shown by the loss of the active p10 caspase-1 sub-unit (Fig. 1Aiii). These results indicate a previously unknown involvement of zinc in this key pro-inflammatory event. Importantly, under these conditions, the production of the pro-inflammatory cytokine IL-6 was not affected by TPEN, suggesting the specificity of the effect (Fig. 1B).
Figure 1. IL-1β secretion from LPS-treated mouse macrophages is zinc dependent. (A) Primary cultured mouse peritoneal macrophages were treated with LPS (1 μg/mL, 2 h) prior to incubation with vehicle (0.5% DMSO) or with the zinc chelator TPEN (10, 50 and 100 μM, 15 min). The inhibitory effects of zinc chelation on ATP-induced (5 mM, 10 min) IL-1β secretion were determined by ELISA (i). Pro-IL-1β processing was determined by Western blot; the 31 kDa band is pro-IL-1β and the 17 kDa band is the caspase-1 cleavage product; cleavage was inhibited by TPEN (ii). Caspase-1 activation was measured by cleavage of the 45 kDa pro-caspase-1 and detection of a 10 kDa sub-unit (iii), which was also inhibited after incubation with the zinc chelator TPEN. (B) The effects of TPEN and ATP on IL-6 release from the experiment described in (A) were determined by ELISA. (C) The effects of TPEN and ATP on cell death from the experiment described in (A) were determined by a LDH assay (i). The protective effects of TPEN on macrophage cell death induced by a 5 min pulse with 5 mM ATP followed by a 25 min incubation were measured by an LDH assay (ii). (D) Changes in FluoZin-3 fluorescence in response to 1 μM ZnPyr applied 1 min before the addition of 10 μM TPEN was determined using the BD Pathway Bioimager (i). Under the conditions described in (A), but with a media change prior to ATP stimulation, the inhibitory effects of TPEN (10 μM, 19 min) on IL-1β secretion can be overcome by coincubation with ZnCl2 (50 μM, 15 min) (ii). IL-1β release from LPS (1 μg/mL, 2 h) and ATP (5 mM, 10 min) treated peritoneal macrophages were not inhibited by the copper chelator TTM or the iron chelator SIH but was abolished by the zinc chelator TPEN (iii). Data show the mean±SD of at least three independent experiments. Blots are representative of three experiments. ***p<0.001.
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In the experiment described in the previous paragraph, macrophages were incubated with ATP for 10 min. We have reported recently that after this period of stimulation the macrophages have released significant quantities of IL-1β without leaking the cytosolic protein lactate dehydrogenase (LDH), which is used as a marker for a loss of membrane integrity 3. Under these conditions ATP did not increase LDH release above control levels and there was no effect of TPEN (Fig. 1Ci). However, stimulation of P2X7-receptor-induced IL-1β release from LPS-primed macrophages does result in macrophage cell death when assayed at later time points, and this is dependent on caspase-1 15. Thus, we hypothesised that TPEN would also block P2X7-receptor-induced cell death. We tested the effects of TPEN on the cell death induced by a 5 min ATP pulse, followed by 25 min incubation in the absence of ATP. We observed that a 15 min pre-incubation with TPEN completely inhibited ATP-induced cell death (Fig. 1Cii). These data suggest therefore that the mechanisms of ATP-induced and caspase-1-dependent cell death and IL-1β release are both regulated by zinc.
We next tested the hypothesis that the inhibitory effects of TPEN on IL-1β secretion would be overcome by the addition of excess zinc. To test this ZnCl2 was added to TPEN-containing macrophages. However, to ensure that the addition of ZnCl2 did not bind TPEN extracellularly, and prevent its cellular accumulation, we performed an experiment to demonstrate TPEN loading. LPS-primed macrophages were loaded with the selective Zn2+ indicator FluoZin-3 16 and imaged on a BD Pathway Bioimager. FluoZin-3-loaded macrophages were incubated with 1 μM of the Zn2+ ionophore, 1-hydroxypyridine-2-thione (zinc salt) (ZnPyr) for 1 min to increase the level of labile zinc (Fig. 1Di). TPEN (10 μM) was then added to the well and a rapid drop in cellular FluoZin-3 fluorescence was observed, returning to baseline within 4 min (Fig. 1Di). This demonstrated that within 4 min of TPEN loading there was sufficient within the cell to abolish a ZnPyr-induced increase in labile zinc. Therefore, ZnCl2 (50 μM) was added to the cells 4 min after the addition of TPEN and incubated for a further 15 min.
After 15 min the media were removed from all wells and fresh media or fresh media containing 5 mM ATP were added for a further 10 min. The supernatants were then analysed for released IL-1β by ELISA. The media change after the TPEN incubation was introduced since μM zinc is known to inhibit P2X7-receptor function 17 and may thus hinder interpretation of data. There was no difference in the levels of IL-1β released from LPS-primed macrophages when treated alone with vehicle, TPEN, ZnCl2 or TPEN plus ZnCl2 (Fig. 1Dii). ATP induced IL-1β release, and this was inhibited by TPEN (Fig. 1Dii). ZnCl2 had no effect on ATP-induced IL-1β release but did rescue the effects of TPEN (Fig. 1Dii). Thus, these data strongly suggest the zinc dependence of ATP-induced IL-1β release.
TPEN binds zinc preferentially, but also binds iron and copper, but not calcium or magnesium ions at biologically relevant levels 18. Therefore, we tested the effects of other selective metal ion chelators. The selective iron chelator, salicylaldehyde isonicotinoyl hydrazone (SIH), and the selective copper chelator, ammonium tetrathiomolybdate (TTM), had no effect on ATP-induced IL-1β secretion from LPS-primed macrophages (Fig. 1Diii), suggesting that the effect of TPEN was due to its binding of zinc.
Increases in intracellular zinc ([Zn2+]i) and the Zn2+ wave (comparable to a Ca2+ wave) have been suggested recently to act as second messengers in immune cells 11. To examine whether this possibility could account for the effects of TPEN described in the previous section, we investigated the effect of P2X7-receptor activation on [Zn2+]i and tested whether manipulation of this influenced IL-1β release. LPS-primed macrophages loaded with FluoZin-3 were imaged for 1 min prior to the addition of vehicle (Fig. 2Ai), 5 mM ATP (Fig. 2Aii) or 1 μM ZnPyr (Fig. 2Aiii). Addition of vehicle had no noticeable effect on [Zn2+]i (Fig. 2Ai). In contrast, stimulation of the P2X7-receptor with 5 mM ATP induced a robust decline from baseline fluorescence (Fig. 2Aii). Incubation with the ionophore ZnPyr caused an increase in [Zn2+]i (Fig. 2Aiii). The FluoZin-3 fluorescence baseline detected in the unstimulated cells likely represents the pool of labile zinc. Manipulation of the labile zinc pool by P2X7-receptor activation in this way may represent an efflux of zinc through the P2X7 channel.
Figure 2. Rapid changes in labile zinc and effects on IL-1β secretion in mouse macrophages. (A) Primary cultured mouse peritoneal macrophages were treated with LPS (1 μg/mL, 2 h), and changes in FluoZin-3 fluorescence in response to vehicle (i), 5 mM ATP (ii) or 1 μM ZnPyr (iii) were determined using the BD Pathway Bioimager. (B) Under the conditions described in (A), FluoZin-3 fluorescence was used to record an increase in the labile zinc pool in response to 1 μM ZnPyr and how this was affected by the addition of vehicle (i), 5 mM ATP (ii) or 50 μM TPEN (iii). (C) Peritoneal macrophages were treated with LPS (1 μg/mL, 2 h)±1 min pre-incubation with 1 μM ZnPyr. The effects of a 5 and 10 min incubation with 5 mM ATP on IL-1β release was measured by ELISA (i), and pro-IL-1β processing was measured by Western blot (ii). ELISA results show the mean±SD of at least three independent experiments. Blots are representative of three experiments.
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We manipulated the labile zinc pool further to investigate how this affects IL-1β processing and release. LPS-primed macrophages, loaded with FluoZin-3, were imaged for 1 min, treated with ZnPyr (1 μM), then imaged for a further minute and subjected to addition of vehicle, ATP (5 mM) or TPEN (50 μM) to assess the effects on FluoZin-3 fluorescence. Addition of vehicle 1 min after the addition of ZnPyr had no effect on [Zn2+]i (Fig. 2Bi). Addition of ATP again induced a rapid drop in [Zn2+]i (Fig. 2Bii), confirming our earlier observation (Fig. 2Aii). TPEN, added 1 min after treatment with ZnPyr, resulted in a rapid drop in cellular fluorescence, confirming that we were indeed manipulating levels of labile zinc (Fig. 2Biii).
These imaging experiments indicate that the changes in [Zn2+]i observed in response to ATP stimulation are unlikely to be important for the activation of caspase-1 because if a drop in [Zn2+]i was important, the effects of ATP on IL-1β release would be similar to that of TPEN. Importantly, we did not observe a rise in FluoZin-3 fluorescence in response to ATP suggesting that we were not inducing a second messenger action of zinc as described previously 11. We also tested the hypothesis that a short (1 min) pre-incubation with ZnPyr would not inhibit ATP-induced processing and secretion of IL-1β. The incubation with ZnPyr would increase cellular labile zinc and buffer its loss after ATP stimulation. We have reported previously that secretion of IL-1β from murine macrophages is minimal after 5 min incubation with ATP (5 mM) but almost complete at 10 min 3. We measured IL-1β release 5 or 10 min after incubation with ATP (5 mM), with and without a 1 min pre-incubation with ZnPyr (1 μM). Secretion of IL-1β after 5 min incubation with ATP was less than that with a 10 min incubation and release at both times was completely unaffected by ZnPyr (Fig. 2Ci). Analysis of the supernatants confirmed that the processing of pro-IL-1β was also unaffected (Fig. 2Cii). The effect of a 10 min incubation with ZnPyr (1 μM) alone is also shown, confirming that IL-1β processing and release is independent of increases in [Zn2+]i (Fig. 2C). Thus, these data suggest that rapid changes in labile zinc do not contribute to ATP-induced caspase-1 activation and IL-1β secretion.
It is possible that TPEN interferes directly with ATP binding to the P2X7-receptor. To address this possibility we investigated the effects of TPEN on IL-1β secretion induced by the K+ ionophore nigericin, which induces caspase-1-dependent secretion of IL-1β independent of the P2X7-receptor 15. Pre-treatment of LPS-primed macrophages with TPEN (50 μM, 15 min) abolished nigericin (20 μM, 10 min) induced secretion of IL-1β (Fig. 3Ai) and processing of pro-IL-1β (Fig. 3Aii), suggesting that TPEN acts independent of the P2X7-receptor.
Figure 3. Zinc-dependent mechanism. (A) Nigericin (20 μM, 10 min)-induced IL-1β release (i) and pro-IL-1β processing (ii) in LPS-treated (1 μg/mL, 2 h) peritoneal macrophages was inhibited by TPEN. (B) In vitro inflammasome assembly and caspase-1 activity, induced by hypotonic lysis of LPS-treated peritoneal macrophages was measured by Ac-YVAD-AMC cleavage, and was not inhibited by TPEN. RFU, relative fluorescence units. (C) The effects of a 20 min incubation of 50 μM TPEN on the Fura-2 (F340/F360), [Ca2+]i response to 1 mM ATP in P2X7 expressing HEK-293 cells (i). Representative fluorescence traces showing the effects of vehicle (DMSO), TPEN (50 μM), and 10panx1 (mimetic pannexin-1 peptide, 400 μM), on ATP (3 mM) induced ethidium dye uptake in P2X7 expressing HEK-293 cells (ii). A summary of the slope of ethidium dye uptake in P2X7 expressing HEK-293 cells (iii). (D) Representative fluorescence traces showing the effects of vehicle (DMSO), TPEN (50 μM), and 10panx1 (mimetic pannexin-1 peptide, 400 μM), on ATP (3 mM) induced ethidium dye uptake in LPS-treated primary mouse peritoneal macrophages (i). A summary of the slope of ethidium dye uptake in primary mouse peritoneal macrophages (ii). Data show the mean±SD of at least three independent experiments. Fluorescence traces are representative of at least three separate experiments. ***p<0.001, **p<0.01.
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The inflammasome constitutes the large protein complex required for the activation of caspase-1 19. Lysis of LPS-primed cells in a hypotonic solution containing little or no K+ enables oligomerisation of the adaptor proteins required to reconstitute a NALP inflammasome in vitro19, 20. Increasing K+ concentrations inhibits inflammasome assembly 20. We used this assay to test whether TPEN had any direct effects on inflammasome assembly, or caspase-1 activity. Cleavage of the fluorescent caspase-1 substrate Ac-YVAD-AMC was used as an indicator for inflammasome assembly. At 4°C no active inflammasomes were formed (Fig. 3B). However, incubation of lysates at 37°C induced a robust activation of caspase-1 activity that was inhibited by 130 mM/K+ as reported previously 20. Co-incubation with TPEN (50 μM) had no effect on caspase-1 activity induced in this assay (Fig. 3B). These data suggest that TPEN is not a direct inhibitor of inflammasome assembly or of caspase-1 activity.
Pannexin-1 is a membrane protein in mammalian cells, which is structurally related to invertebrate hemichannel proteins and is known to be essential for both ATP- and nigericin-induced caspase-1 activation and IL-1β secretion 21, 22. We tested whether pannexin-1 activity was zinc-dependent. The nigericin data suggested that the zinc-dependent effect could be independent of P2X7-receptor activation (Fig. 3A). We confirmed that TPEN did not inhibit P2X7-receptor activation by measuring changes in [Ca2+]i in response to 1 mM ATP in vehicle and TPEN (50 μM, 15 min) loaded human embryonic kidney (HEK)-293 cells over-expressing the mouse P2X7-receptor, and loaded with Fura-2 (Fig. 3Ci). Pannexin-1 is essential for a component of the dye uptake pathway induced by P2X7-receptor activation since RNAi knockdown, or inhibition by a mimetic peptide (10panx1) delays dye uptake rather than abolish it 22. We found that a short incubation (20 min) with TPEN (50 μM) inhibited ATP-induced ethidium dye uptake to the same extent as incubation with the pannexin-1 mimetic peptide 10panx1 in the P2X7 expressing HEK-293 cells (Fig. 3Cii and iii).
These data suggested that the zinc-dependent step was the activation of pannexin-1. P2X7-receptor over-expressing HEK-293 cells provide a robust model for investigating P2X7, and pannexin-1-dependent mechanisms 21, 22 and we confirmed that the effect was also present in LPS-treated primary mouse peritoneal macrophages (Fig. 3Di and ii).