Release of Interleukin-1α or Interleukin-1β Depends on Mechanism of Cell Death^*

Materials

RPMI 1640 medium and DMEM, fetal bovine serum (FBS), glutamine, and a streptomycin/penicillin antibiotic solution were all purchased from Invitrogen. Bacterial lipopolysaccharide (LPS, Escherichia coli 026:B6) and staurosporine (STS)² were purchased from Sigma. Ac-YVAD-CHO, IETD-CHO, cycloheximide (CHX), ALLN, calpain inhibitor III, EST, and PD150606 were purchased from Merck Chemicals, Ltd. CHX causes apoptosis by inhibiting protein translation and subsequently cell growth, whereas STS is a broad spectrum kinase inhibitor that induces apoptosis. Z-VAD-fmk was purchased from Promega. Necrostatin-1 was purchased from Sigma. The anti-mouse IL-1β and IL-1α antibodies were from R&D Systems. Secondary antibody HRP conjugates were from DAKO.

Mice

NLRP3^−/− and ASC^−/− mice were generously provided by Dr. Vishva Dixit, Genentech (18, 19). C57BL/6J mice were purchased from Harlan UK.

Cell Culture

Bone marrow derived macrophages (BMDMs) were derived from the bone marrow of adult, male C57BL/6 mice as described previously (20). Cells were cultured in DMEM supplemented with 10% FCS, 100 units/ml penicillin and 100 μg/ml streptomycin, and 30% L929 supernatant containing macrophage-stimulating factor. After 6–9 days, the resulting BMDMs were plated at a density of 5 × 10⁵ cells/well and treated with LPS (1 μg/ml, 4 h) prior to incubation with apoptosis inducers (or other treatments as indicated) for 0.5–24 h as indicated.

Primary peritoneal macrophages were prepared from adult, male C57BL/6 mice, as described previously except no HEPES was added to the medium (21). Peritoneal macrophages were cultured at a density of 1 × 10⁶ cells/ml in RPMI 1640 medium supplemented with 5% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin. 24 h after culture cells were treated with LPS (1 μg/ml, 2 h) prior to a 4-h incubation with 100 μm Z-VAD (or other treatments where indicated). Supernatants and lysates were harvested for subsequent analyses. For the experiment presented in Fig. 6 the “calcium-containing” buffer was composed of (in mm): NaCl (121), KCl (5.4), MgCl₂ (0.8), CaCl₂ (1.8), NaHCO₃ (6), HEPES (25), and glucose (5). The “calcium-free” buffer was composed of (in mm): NaCl (121), KCl (5.4), MgCl₂ (0.8), NaHCO₃ (6), HEPES (25), glucose (5), and EGTA (5).

Western Blotting

Supernatants and lysates were harvested and prepared in sample buffer containing 1% β-mercaptoethanol. Samples were boiled and then electrophoresed on 12% SDS-acrylamide gels. Proteins were transferred to a nitrocellulose membrane and blotted with primary antibodies, followed by HRP-conjugated secondary antibodies, and subsequent exposure using enhanced chemiluminesence reagents (ECL; Amersham Biosciences).

Detection of IL-1α and IL-1β by ELISA

Measurement of IL-1α and IL-1β released into macrophage culture supernatants was done using specific mouse IL-1β and IL-1α ELISA kits (R&D Systems) following the manufacturer's instructions.

Cell Death Assays

Cell death was recorded by measuring the release of the enzyme lactate dehydrogenase (LDH) from the cells using the CytoTox-96 assay (Promega) according to manufacturer's instructions. Cell death data are either presented as percentage LDH release compared with a Tritonlysis control or as a -fold increase over LDH release in vehicle-treated samples. Apoptosis was measured using a luminogenic Caspase-Glo 3/7 reagent according to the manufacturer's instructions (Promega).

Data Analysis

Data are presented as the mean ± S.D. of at least three separate cultures. Groups of data were analyzed by one-way analysis of variance followed by Bonferroni's multiple comparison test. Statistical significance was assumed when p < 0.05. All Western blots presented are representative of three independent experiments from three separate cultures.

Apoptotic Stimuli Induce IL-1 Release

BMDMs were incubated with or without LPS (1 μg/ml, 4 h) before a media change into serum-free conditions and incubation with the apoptotic inducer CHX (100 μm) or STS (5 μm), for between 0.5 and 24 h. Supernatants were collected at the indicated times and analyzed for cell death (percentage LDH release, Fig. 1A), IL-1β release (Fig. 1B), or IL-1α release (Fig. 1C). In a parallel experiment apoptosis was assessed using a luminogenic caspase-3/7 substrate (Fig. 1D). Incubation with both CHX and STS was toxic, causing an early induction of apoptosis which tapered off as the cells underwent secondary necrosis and released LDH, and this was not affected by LPS priming (Fig. 1, A and D). CHX and STS also induced the release of IL-1β (Fig. 1B) and IL-1α (Fig. 1C). However, this occurred only in LPS-primed cells. Thus, using two classic inducers of apoptosis we were able to stimulate the release of both IL-1β and IL-1α from LPS-primed BMDMs.

Apoptosis-induced IL-1β Release Is Caspase-8-dependent

Apoptosis has been reported to activate the NLRP3 inflammasome/caspase-1 to induce IL-1β release (15) and also by inflammasome-independent but caspase-8-dependent mechanisms (16, 17). Thus, we sought to determine the mechanisms regulating CHX- and STS-induced IL-1β and IL-1α release from LPS-primed BMDMs. BMDMs isolated from WT, NLRP3^−/−, or ASC^−/− mice were treated with LPS (1 μg/ml, 4 h) and then treated with CHX (100 μm) or STS (5 μm) in serum-free medium for 24 h because we had established that at this time robust death and IL-1 release were observed (Fig. 1). Cell death in response to CHX or STS was unaffected in NLRP3^−/− or ASC^−/− compared with WT (Fig. 2A). Furthermore, cell death measured by LDH release in response to CHX or STS was also unaffected by incubation with the selective caspase-8 inhibitor IETD (Fig. 2A). CHX- or STS-induced IL-1β processing and release were partially inhibited by the loss of NLRP3 (p < 0.001) or ASC (p < 0.01 (CHX), p < 0.001 (STS)) compared with WT and were completely inhibited by IETD (Fig. 2B). Importantly, IETD also inhibited apoptosis induced by CHX measured using caspase-3/7 activation (Fig. 2D). CHX and STS induced significant IL-1α processing and release (p < 0.01) compared with vehicle treatment, and this was not affected by inflammasome knockout or by caspase-8 inhibition (Fig. 2C). Thus, these data suggest that apoptotic stimuli induced a caspase-8-dependent apoptosis and regulation of IL-1β processing and release, with a partial involvement of the NLRP3 inflammasome, whereas release of IL-1α followed necrotic cell death.

Our data above suggested only a partial role for caspase-1 in the processing and release of IL-1β in response to CHX and STS. This was confirmed using the caspase-1 inhibitor YVAD (100 μm) which barely affected STS- or CHX-induced IL-1β release and had no effect on cell death or IL-1α release, in LPS-primed BMDMs (Fig. 3A). Incubation of LPS-primed BMDMs with the pan-caspase inhibitor Z-VAD (100 μm) completely inhibited STS- and CHX-induced IL-1β processing and release, but had no effect on STS- or CHX-induced cell death or IL-1α release (Fig. 3B). Whereas the caspase-8 inhibitor IETD and the caspase-1 inhibitor YVAD had no effect on cell death alone when incubated with LPS-primed BMDMs (Figs. 2A and 3A), Z-VAD induced cell death and significant IL-1α processing and release (Fig. 3B).

Necroptosis Induces IL-1α Release

Treatment of macrophages with LPS and the pan-caspase inhibitor Z-VAD induces RIP3-dependent necroptosis (22), a caspase-independent form of programmed necrosis. Necroptosis can be inhibited by the RIP1 inhibitor necrostatin-1 (NEC1) (23, 24). Thus, the data we present above (Fig. 3B) suggested that in our LPS-primed BMDMs, Z-VAD induced necroptosis, that this drove IL-1α processing and release, and that this was completely independent of IL-1β. To further confirm that necroptosis was regulating the processing and release of IL-1α we incubated LPS-primed BMDMs with Z-VAD (100 μm) in the absence or presence of the RIP1 inhibitor NEC1 (100 μm). To determine whether necroptotic responses to LPS and Z-VAD were inflammasome-dependent we also repeated the LPS and Z-VAD experiment in BMDMs isolated from NLRP3^−/− and ASC^−/− mice (Fig. 4). Z-VAD induced cell death in LPS-primed BMDMs from WT, NLRP3^−/−, and ASC^−/− mice, and this was not significantly affected by NEC1 (Fig. 4A). In LPS-primed BMDMs from all strains Z-VAD induced IL-1α release, and this was significantly inhibited by NEC1 (Fig. 4C). There was no release of IL-1β over vehicle treatment (Fig. 4B). Interestingly, the release of IL-1α from LPS- and Z-VAD-treated ASC^−/− cells was lower than from WT or NLRP3^−/− cells, suggesting that under these conditions release of IL-1α may be partially ASC-dependent (Fig. 4C). We repeated these experiments in primary mouse peritoneal macrophages, another cell type commonly used to interrogate mechanisms of IL-1 release. In LPS-primed primary peritoneal macrophages, NEC1 (100 μm) inhibited Z-VAD-induced cell death (100 μm, 4-h treatment) (Fig. 5A) and processing and release of IL-1α (Fig. 5C). Again, under these conditions, release of IL-1β did not occur (Fig. 5B). These data suggest that necroptosis regulates the release of IL-1α independently of the inflammasome and IL-1β.

Necroptosis-induced IL-1α Release Is Ca²⁺- and Calpain-dependent

Pro-IL-1α is believed to be cleaved by calcium (Ca²⁺)-dependent proteases of the calpain family (8, 9). To determine the nature of IL-1α processing during necroptosis we first stimulated LPS-primed BMDMs with Z-VAD (100 μm) in Ca²⁺-containing or Ca²⁺-free medium (Fig. 6). Incubation of the cells in Ca²⁺-free medium (24 h) alone induced cell death comparable with Z-VAD treatment (Fig. 6A) and was also a sufficient trigger for IL-1β processing and release (Fig. 6B). LPS-primed cells incubated in Ca²⁺-free conditions in the presence of Z-VAD did not secrete IL-1β (Fig. 6B). In contrast, Z-VAD induced IL-1α processing and release in Ca²⁺-containing medium, and this was inhibited in Ca²⁺-free conditions (Fig. 6C). These data suggest that the processing and release of IL-1α during necroptosis are Ca²⁺-dependent.

An anti-calpain I antibody blocked Ca²⁺-dependent processing of pro-IL-1α in lysates U937 histiocytic lymphoma cells (25). We therefore tested a panel of calpain I inhibitors against the effects of Z-VAD in LPS-primed BMDMs (Fig. 7). Over the course of this experiment several of the calpain inhibitors caused cell death, and Z-VAD-induced cell death was not inhibited by calpain inhibition (Fig. 7A). Related to its effects on cell death the calpain inhibitor ALLN (100 μm) induced some IL-1β release (Fig. 7B). This was not related to necroptosis however because it was inhibited by Z-VAD (Fig. 7B). Furthermore, none of the calpain inhibitors tested induced levels of IL-1α release comparable with Z-VAD (Fig. 7C). ALLN (100 μm), calpain inhibitor III (MDL-28170, 100 μm), and calpeptin (100 μm) significantly inhibited Z-VAD-induced IL-1α release (Fig. 7C). Together these data suggest that necroptosis drives the processing and release of IL-1α through a mechanism that is Ca²⁺-dependent and at least partially dependent upon calpains.