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and IL-1
from Murine Macrophages1
School of Biological Sciences, University of Manchester, Manchester, United Kingdom
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Abstract |
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and IL-1
) present in the cytosol of activated monocytes/macrophages. IL-1 is synthesized as an inactive precursor that lacks a signal sequence, and its trafficking does not use the classical endoplasmic reticulum-Golgi route of secretion. Using primary cultured murine peritoneal macrophages, we demonstrate that P2X7 receptor activation causes release of IL-1 and IL-1 via a common pathway, dependent upon the release of Ca2+ from endoplasmic reticulum stores and caspase-1 activity. Increases in intracellular Ca2+ alone do not promote IL-1 secretion because a concomitant efflux of K+ through the plasmalemma is required. In addition, we demonstrate the existence of an alternative pathway for the secretion of IL-1, independent of P2X7 receptor activation, but dependent upon Ca2+ influx. The identification of these mechanisms provides insight into the mechanism of IL-1 secretion, and may lead to the identification of targets for the therapeutic modulation of IL-1 action in inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1, believed to exert identical actions (1). Both IL-1 isoforms are synthesized as 31-kDa precursors (pro-IL-1). Pro-IL-1, but not pro-IL-1, is biologically active (1). Pro-IL-1 must be cleaved to its mature, active 17-kDa form by the enzyme caspase-1 (2). The mechanism of cellular release remains largely unknown: the IL-1 precursor molecules are synthesized without a signal sequence, and their trafficking does not use the classical endoplasmic reticulum (ER)3-Golgi route (3).
Synthesis of pro-IL-1 in cultured monocytes and macrophages is commonly induced by inflammatory stimuli, such as LPS (4, 5). LPS is not sufficient to promote IL-1 secretion in cultured cells; an additional stimulus is required. Extracellular ATP induces the release of mature IL-1 from LPS-primed macrophages via activation of the P2X7 receptor (6). P2X7 receptor stimulation promotes caspase-1 activation that induces cell death, independently of its IL-1-processing properties (7). Caspase-1-mediated cell death and IL-1 release by activated macrophages are reported in a number of acute inflammatory conditions (8, 9).
The actions of extracellular ATP are mediated via membrane receptors belonging to two purinergic receptor families, the ionotropic P2X receptors and the metabotropic P2Y receptors. Both receptor types are expressed by immunocompetent cells (10, 11, 12, 13). Studies in activated macrophages and monocytes using Ca2+ and K+ ionophores indicate that Ca2+ influx has no effect on IL-1 processing and release, but that agents capable of depleting cellular K+, including hypotonic conditions, promote mature IL-1 secretion (4, 14, 15). These studies suggest that K+ movement or local concentration influences the activity of caspase-1 directly or indirectly (15). In addition, K+ leakage from human monocytes activates Ca2+-independent phospholipase A2, which is essential for IL-1 maturation and release, while activation of Ca2+-dependent phospholipase A2 by the Ca2+ ionophore A23187 inhibits IL-1 maturation (16). Taken together, these data suggest that maturation and release of IL-1 are dependent on K+ efflux from the cell, and that Ca2+ has inhibitory effects on IL-1 secretion.
Processing of IL-1 requires Ca2+-dependent calpain activity (17, 18, 19). The source of Ca2+ required for calpain activation and subsequent IL-1 processing and release is extracellular, as calcium ionophores induce the release of mature IL-1 (17, 19), a process inhibited by the Ca2+ chelator EGTA in the extracellular medium (18). ATP-induced macrophage cell death is accompanied by the processing and release of both IL-1 and IL-1, and their release has been suggested to occur via a similar mechanism (20).
The role of intracellular Ca2+ ([Ca2+]i) in IL-1 secretion has not been investigated. The purpose of this investigation was to examine the effects of increased [Ca2+]i on the secretion of IL-1. We demonstrate in this study that ATP-induced IL-1 release from LPS-primed murine peritoneal macrophages is largely independent of Ca2+ influx, but depends on Ca2+ release from intracellular stores. This release is necessary, but not sufficient to promote IL-1 release, as it also requires an efflux of K+. Caspase-1 is an essential component in this pathway and participates in ATP-induced IL-1 secretion. Independently of P2X7 receptor activation, a separate mechanism dependent on Ca2+ influx exists for the secretion of IL-1. The discovery that Ca2+ is important for IL-1 secretion provides a useful target for the modulation of IL-1 release from macrophages.
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Materials and Methods |
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Adult male C57BL/6 in wild type (WT) or caspase-1 knockout (KO) mice (25 g) were sacrificed by rising CO2 concentrations in accordance with Home Office (U.K.) procedures, and the peritoneal cavity was lavaged with 8 ml RPMI 1640 medium (containing 25 mM HEPES, pH 7.3, 2 mM glutamine, and 5% FCS (Invitrogen Life Technologies, Paisley, U.K.)). The medium recovered from four to five mice was pooled, and cells were collected by centrifugation (80 x g, 10 min) and plated in 24-well plates at a density of 0.5 x 106 cells/well. The macrophages were allowed to adhere for 2 h (37°C, 5% CO2), washed with fresh medium to remove unattached cells, and incubated overnight. The genetic status of the caspase-1 KO mice was confirmed by PCR using the following primers: CCT GAG GGC AAA GAG GAA GC and GAG CAG AAA GCAATA AAA TC (21).
Cells were primed for 2 h with 1 µg/ml LPS (Escherichia coli 026:B6; Sigma-Aldrich, Poole, U.K.). After 2 h, cells were subjected to a 5-min pulse with 1or 5 mM ATP, followed by a further 10 or 25 min in culture before supernatants and lysate were harvested. Modifications to specific experiments are indicated in the text where appropriate.
To examine the role of Ca2+ in ATP-induced IL-1 release, LPS-primed macrophages were incubated with the cell-permeable Ca2+-chelator BAPTA-AM (Sigma-Aldrich) for 15 min, followed by 15-min incubation in normal medium (RPMI 1640) to allow de-esterification. The cells were then pulsed with 1 mM ATP or vehicle (H2O) for 5 min, followed by a further incubation in RPMI 1640 for 10 min, before the supernatants were removed.
For Ca2+-free experiments, LPS-primed macrophages were incubated for 5 min with 1 mM ATP in RPMI 1640 medium and in PBS minus Ca2+ (Ca2+ free). Cells were then incubated for a further 10 min in RPMI 1640 medium before IL-1 and cell death analysis.
Reagents (e.g., LPS, ATP, BAPTA-AM, ionomycin, nigericin, and thapsigargin) were purchased from Sigma-Aldrich. Fura 2-acetoxymethyl ester was obtained from Molecular Probes (Eugene, OR).
Detection of IL-1 release by ELISA
Release of IL-1 (both and ) into the culture medium in response to LPS and/or ATP was measured by specific mouse sandwich ELISAs, generously provided by S. Poole of the National Institute for Biological Standards and Control (NIBSC, Potters Bar, U.K.). The assays are specific for each cytokine, with no cross-reaction with other cytokines. The levels of detection were 
20 pg/ml, and internal quality controls were included in each assay. A limitation of this assay is its inability to distinguish mature from pro-IL-1.
Detection of IL-1 by immunoblot
Pro- or mature IL-1 in macrophage lysate (10 µg protein) or released into the supernatant (10 µl) was detected by immunoblot analysis. Proteins were resolved on a 12% SDS-polyacrylamide gel and subsequently transferred onto a nitrocellulose membrane (Amersham, Little Chalfont, U.K.). To reduce nonspecific binding of Ab, the membrane was washed in 5% low fat milk (in PBS-(0.1%) Tween) at room temperature for 1 h. The membranes were then probed with a polyclonal Ab raised against mouse IL-1 (S329, NIBSC; 1/1000 dilution) or mouse IL-1 (S113B, NIBSC; 1/1000 dilution), followed by a polyclonal Ab raised against sheep IgG, conjugated to HRP (NIBSC; 1/4000 dilution), and detection by ECL (Amersham). The Ab dilutions were prepared in a 5% low fat milk/PBS-Tween (0.1%) solution.
Cytotoxicity of LPS and ATP
The cytotoxic effects of ATP and LPS were assessed by the CytoTox 96 assay (Promega, Madison, WI), which measures lactate dehydrogenase (LDH) release from dying cells. The assay was performed following the manufacturer's instructions. Values were expressed as percentage of total LDH release, obtained by adding Triton X-100 (9% v/v, final concentration) to untreated cells.
[Ca2+]i recordings
All solutions were prepared freshly from stock solutions kept at 4°C. Cultured macrophages were loaded with the Ca2+ indicator fura 2-AM by incubating adherent cells on glass coverslips in normal physiological bathing solution (NaCl, 150 mM; KCl, 5.4 mM; CaCl2, 2 mM; MgCl2, 1 mM; HEPES/NaOH, 10 mM; glucose, 10 mM; pH 7.4), supplemented with 5 µM fura 2-acetoxymethyl ester for 20 min at room temperature. The cells were then washed twice with physiological saline and stored in the dark for 30 min. Fluorescence images (at the emission wavelength 530 ± 10 nm) were captured using a Olympus IX70 inverted microscope (x40 UV objective) equipped with a charge-coupled device cooled intensified camera (Pentamax Gene IV; Roper Scientific, Marlow, U.K.). The specimen was alternately illuminated at 340 and 380 nm by a monochromator (Polychrom IV; T.I.L.L. Photonics, Martinsreid, Germany) at a cycle frequency 1–2 Hz. Control of the experiment and off-line analysis were performed by use of MetaFluor/MetaMorph software (Universal Imaging, Downingtown, PA) running on a Windows 98 workstation.
The [Ca2+]i was calculated from the ratio (R) of fluorescence recorded at 340 and 380 nm excitation wavelengths (22): [Ca2+]i = Kd (R - Rmin)/ (Rmax - R), where Rmin is the fluorescence ratio of Ca2+-free fura 2, and Rmax is the ratio of Ca2+-bound fura 2. The constant Kd was determined empirically. The system was calibrated in situ by using an ionomycin-based intracellular calibration procedure, as described previously (23), and the Rmin, Rmax, and Kd were 0.2, 2.8, and 437 nM, respectively.
Data analysis
The data are presented as the mean ± SEM of triplicate determinations from at least three separate cultures. Statistical significance was assessed by one-way ANOVA, followed by the Newman-Kuels multiple comparison tests. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Results |
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release
The aim of the first experiment was to establish whether [Ca2+]i is involved in ATP-induced IL-1 release. Fig. 1A demonstrates that ATP-induced IL-1 release from LPS-primed macrophages was significantly inhibited by preincubation of the cells with 50 or 100 µM of the membrane-permeable Ca2+ chelator, BAPTA-AM (Fig. 1A, p < 0.001). Immunoblot analysis showed that preincubation of the macrophages with BAPTA-AM inhibited the appearance of mature, 17-kDa caspase-1-generated IL-1 in the supernatant, and also inhibited the intracellular processing of pro- to mature IL-1 (Fig. 1B). The increase in pro-IL-1 release after application of higher doses of BAPTA-AM (Fig. 1B, lower panel) was probably caused by the increase in cell death observed at these concentrations (Fig. 1C), and not by secretion of immature IL-1. Basal macrophage cell death, measured by LDH release, was increased by ATP (from 4% to 23 ± 5% of total LDH), and was not affected by preincubation of the macrophages with BAPTA-AM (10–100 µM; Fig. 1C). The decrease in mature, secreted IL-1 further supports the concept that the processing and release of IL-1 are closely linked. Thus, the results of the BAPTA-AM experiment show that increases in [Ca2+]i are important for ATP-induced IL-1 processing and release.
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processing and release, LPS-primed macrophages were stimulated with ATP in the presence and absence of extracellular Ca2+. ATP-induced IL-1 release was not significantly altered in the absence or presence of extracellular Ca2+ when compared with normal conditions (from 2580 ± 680 to 5410 ± 1400 pg/ml in PBS minus Ca2+ (NS) and 2600 ± 1100 pg/ml in PBS plus 1 mM Ca2+; Fig. 2A). The absence of Ca2+ in the extracellular medium did not affect the release of processed IL-1 (Fig. 2B). ATP-induced cell death still occurred in the absence of extracellular Ca2+, and was significantly greater (52 ± 4% of total LDH) than the response to ATP under normal conditions (17% of total LDH, p < 0.001; Fig. 2C). The readdition of 1 mM Ca2+ to the Ca2+-free medium before the ATP pulse significantly reduced the ATP-induced cytotoxicity (32 ± 4% of total LDH, p < 0.01 compared with ATP in PBS minus Ca2+). These results indicate that in the absence of extracellular Ca2+, ATP is a more potent agonist of the P2X7 receptor (24), and that influx of extracellular Ca2+ is not essential for ATP-induced processing and release of IL-1.
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(2160 ± 150 pg/ml, p < 0.01 vs vehicle; Fig. 3A) that correlated with significant cell death (64 ± 5% of total LDH, p < 0.001; Fig. 3C). In response to ionomycin, IL-1 was released exclusively as the pro form, suggesting nonspecific release resulting from cell death (Fig. 3B). The K+ ionophore nigericin (20 µM) also induced significant IL-1 release (3160 ± 500 pg/ml, p < 0.001), mainly in the mature form (Fig. 3B). The nigericin-induced IL-1 processing and release were dependent on [Ca2+]i, as preincubation of the macrophages with BAPTA-AM (50 µM) significantly inhibited release of mature IL-1 (from 3160 ± 500 to 1380 ± 570 pg/ml, p < 0.01; Fig. 3, A and B). These experiments suggest nigericin affects [Ca2+]i and, like ATP, changes in [Ca2+]i are essential for nigericin-induced IL-1 processing and release.
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processing and release, agents that promote increases in [Ca2+]i, such as 100 µM ATP (subP2X7 activating) and thapsigargin (1 µM), were used. Neither ATP (100 µM) nor thapsigargin (1 µM) induced the release of IL-1 (Fig. 4Ai), and neither were toxic (Fig. 4Bi). However, when macrophages were incubated with thapsigargin (1 µM) for 10 min before a 5-min pulse of ATP (1 mM), or a 15-min incubation with nigericin (20 µM), release of IL-1 was significantly reduced (by 50% for ATP and by 42% for nigericin, respectively, both p < 0.05; Fig. 4Aii). In an attempt to fully deplete [Ca2+]i, 100 µM ATP was coapplied with thapsigargin (1 µM). This further reduced IL-1 release in response to 1 mM ATP (70%, p < 0.01), but had no significant effect on nigericin-induced IL-1 release in thapsigargin-treated cells (Fig. 4Aii). Thus, a significant component of ATP- and nigericin-induced IL-1 release is dependent on the release of Ca2+ from thapsigargin-sensitive [Ca2+]i stores.
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release (21). In response to a 5-min pulse with 5 mM ATP or 15-min incubation with 20 µM nigericin, IL-1 release was increased in LPS-primed macrophages from WT mice, but was completely absent from LPS-primed macrophages from caspase-1 KO mice (Fig. 5, A and B).
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release with changes in [Ca2+]i induced by the pharmacological agents used, [Ca2+]i was imaged from cultured macrophages loaded with the Ca2+-sensitive probe, fura 2. Brief application of 100 µM ATP, 1 mM ATP, or 20 µM nigericin resulted in a rapid, transient [Ca2+]i elevation (Fig. 6, A and B). Importantly, the [Ca2+]i transients induced by 100 µM ATP were only slightly smaller than those induced by 1 mM ATP (Fig. 6, B and C). Thapsigargin, used earlier to examine the effect of increasing [Ca2+]i alone on IL-1 release (Fig. 4Ai), was again applied to the macrophages, this time to examine the effects of prior depletion of the ER calcium store on ATP- and nigericin-induced IL-1 release. Application of thapsigargin resulted in a slow increase in [Ca2+]i because of the unopposed Ca2+ leakage from ER stores. When the ER-Ca2+ stores were depleted, both 100 µM and 1 mM ATP still triggered [Ca2+]i elevation, although its amplitude was substantially reduced (
50%). The [Ca2+]i response to nigericin in thapsigargin-treated cells was reduced by almost 90% (Fig. 6, B and C). These data are consistent with the changes in IL-1 release (Fig. 4), and indicate that both nigericin and ATP induce Ca2+ release from ER stores.
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release
In response to ATP, the release of IL-1 was also Ca2+ dependent (Fig. 7). ATP induced a significant release of IL-1 (2325 ± 510 pg/ml, p < 0.001) that was significantly reduced by preincubation of the cells with 50 µM BAPTA-AM (466 ± 85 pg/ml, p < 0.001 vs ATP alone; Fig. 7Ai). ATP induced the release of both pro- and mature IL-1, and their appearance in the supernatant was inhibited by BAPTA-AM (Fig. 7Aiii). The Ca2+ ionophore ionomycin (2600 ± 150 pg/ml, p < 0.001) and the K+ ionophore nigericin (2215 ± 140 pg/ml, p < 0.001) also induced the release of significant levels of IL-1 (Fig. 7Bi), in both the pro- and mature form (Fig. 7Bii). The intracellular form of IL-1 detected in LPS-primed macrophage cell lysate was the 35-kDa pro form (Fig. 7Aii), indicating that ATP, ionomycin, and nigericin induce calpain activity.
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, the release of IL-1 in response to ATP depends on caspase-1, as ATP-induced IL-1 release was absent in LPS-primed caspase-1 KO macrophages (Fig. 8A). To determine whether caspase-1 is important for the release of IL-1 in response to the Ca2+ and K+ ionophores observed earlier (Fig. 7), LPS-primed caspase-1 KO macrophages were incubated with ionomycin and nigericin. In this experiment, the release of IL-1 in response to nigericin was inhibited, but ionomycin induced significant release of IL-1 (1760 ± 200, p < 0.001; Fig. 8). Nigericin-induced cell death is also caspase-1 dependent, as LDH release in response to nigericin in WT macrophages (46 ± 2% of total LDH) is significantly greater than in caspase-1 KO macrophages (13 ± 1% of total LDH, p < 0.001; Fig. 8C). The cell death that occurs following ionomycin is present in both WT and caspase-1 KO macrophages (Fig. 8C).
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Discussion |
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Previous reports suggest that release of mature IL-1 from activated macrophages and monocytes is dependent on K+ efflux, but independent of Ca2+ fluxes (4, 14, 15). However, we demonstrate in this study for the first time that processing and release of IL-1 depend on the release of Ca2+ from intracellular ER stores. Inhibition of IL-1 release by the membrane-permeable Ca2+ chelator, BAPTA-AM, provides the first evidence for an involvement of [Ca2+]i in IL-1 processing and release. BAPTA-AM did cause modest cell death that was significant at higher concentrations, but this is highly unlikely to have contributed to its inhibition of IL-1 release, because it did not modify IL-1 expression and had opposing actions to ATP, which also causes cell death. In addition, removal of Ca2+ from the extracellular medium potentiated the effect of ATP on IL-1 release and cell death in macrophages. This is consistent with earlier reports, which showed that removal of extracellular Ca2+ enhanced the effects of ATP at the P2X7 receptor (the active ligand is ATP4-) (24), although there is evidence to suggest that the inhibitory effect of extracellular Ca2+ could be allosteric modulation of the P2X7 receptor (26). These results indicate that release of mature IL-1 is dependent on Ca2+ release from intracellular stores rather than an influx of extracellular Ca2+. This is in contrast to a recently proposed mechanism of secretion through microvesicles, shed from the membrane after P2X7 receptor activation in a monocytic cell line. This latter process was found to be dependent on the presence of Ca2+ in the extracellular medium (27).
It is not known how K+ ionophores such as nigericin induce Ca2+-dependent IL-1 processing and release. However, intracellular alkalinization, induced by nigericin or ammonium chloride (NH4Cl), triggers the release of Ca2+ from intracellular stores in a number of systems (28, 29, 30). The data presented in this work demonstrate that nigericin causes increased [Ca2+]i and mature IL-1 secretion in macrophages. This effect is substantially reduced by prior Ca2+ store depletion with the ER Ca2+-ATPase inhibitor, thapsigargin, suggesting that the main source for nigericin-dependent increases in [Ca2+]i is [Ca2+]i stores. Prior incubation of the macrophages with thapsigargin also reduced the ATP-induced [Ca2+]i response (Fig. 6) and IL-1 secretion (Fig. 4Aii), again demonstrating that in response to ATP, IL-1 release is, to a large extent, dependent upon the release of Ca2+ from intracellular stores.
Release of Ca2+ from intracellular stores alone was not sufficient to trigger mature IL-1 release. For example, agents capable of inducing [Ca2+]i transients such as thapsigargin, 100 µM ATP (Fig. 6), and the purely metabotropic stimulus platelet-activating factor (data not shown) failed to induce IL-1 release. In addition, treatment of the cells with ionomycin correlated with a high level of cell death that was accompanied by the release of only pro-IL-1, and is consistent with previous studies (14, 16).
K+ efflux through the P2X7 receptor is well established, but this ion channel is not known to couple with [Ca2+]i stores (31), and it seems unlikely that ATP could induce intracellular store Ca2+ release via this receptor. The present data provide several pieces of evidence to suggest that ATP may activate both ionotropic and metabotropic purinergic receptors, leading to concomitant K+ efflux and Ca2+ release from ER stores (Fig. 9). In addition to P2X7 receptors and other ionotropic P2X receptors (P2X1-6), murine peritoneal macrophages express metabotropic P2Y receptors, activation of which results in inositol-1,4,5-triphosphate generation and release of Ca2+ from intracellular stores (32). Addition of 1 mM ATP to murine macrophages would therefore activate P2X7 receptors, P2Y receptors, and any other P2X receptors present. The Ca2+-imaging experiments suggest an involvement of P2Y receptors by demonstrating that a substantial part of the ATP-induced [Ca2+]i elevation is associated with thapsigargin-sensitive ER Ca2+ stores. The activation of P2Y and P2X receptors by 100 µM ATP (a saturating concentration for all types of purinergic receptor, except the P2X7 receptor) induces a rise in [Ca2+]i almost as great as that induced by 1 mM ATP, but does not induce IL-1 release. Therefore, the critical component of P2X7 receptor activation required for IL-1 release is most likely associated with K+ efflux, while concomitant P2Y receptor activation accounts for the release of Ca2+ from intracellular stores and an increase in [Ca2+]i. The source of the Ca2+ is mainly ER derived, as ATP-induced IL-1 release is not affected by Ca2+ influx through the plasmalemma, but is markedly inhibited by ER store depletion. Thus, the processing and release of IL-1 induced by ATP may be regulated by activation of two distinct classes of purinergic receptors, the metabotropic P2Y receptors, which trigger inositol-1,4,5-triphosphate-induced Ca2+ release from the ER stores, and the P2X7 receptor, which mediates K+ efflux (Fig. 9). Individually, Ca2+ release and K+ efflux are necessary, but not sufficient for IL-1 release; only their simultaneous activation induces the secretion of mature IL-1.
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in response to ATP is also dependent on caspase-1 (Fig. 5). It is unlikely that Ca2+ modulates caspase-1 activity directly, as the caspase-1-dependent cell death in response to ATP (7) is still present in the BAPTA-AM-treated macrophages (Fig. 1C).
The mechanism of IL-1 release is the subject of some controversy. A recently proposed mechanism involves extracellular Ca2+-dependent microvesicle shedding following P2X7 receptor activation of THP.1 monocytes (27). However, in the primary murine macrophages studied in this work, this mechanism appears unlikely to account for IL-1 release, as the vesicular IL-1 would not be accessible to our method of detection, and as IL-1 release was found to be inhibited in the absence of extracellular Ca2+. In human monocytes, 5–10% of cellular IL-1 has been described to colocalize with endolysosomal vesicles (33). However, in this and a previous study, we found that almost 100% of cellular IL-1 is secreted in response to ATP (7). Thus, our data suggest that IL-1 may be either secreted by another mechanism, or that the increase in [Ca2+]i is critical for an as yet unknown aspect of IL-1 packaging/vesicle fusion.
The processing and release of mature IL-1 from macrophages and monocytes have been studied far less than that of IL-1. However, significant differences in the processing of these cytokines exist. Processing of pro-IL-1 depends on Ca2+-dependent calpain enzymes (17, 18, 19). Although extracellular Ca2+ was demonstrated to be unimportant for the secretion of mature IL-1, Ca2+ ionophores such as ionomycin induce the release of mature IL-1 (17, 19). The source of Ca2+ required for calpain activation in IL-1 processing and release is thus thought to be extracellular, as this process is inhibited by the presence of EGTA in the extracellular medium (18). The data presented in this work on the effects of ionomycin are consistent with these early reports, and demonstrate that ionomycin can induce the release of mature IL-1 (Fig. 7). These experiments also demonstrate that the K+ ionophore nigericin induces release of mature IL-1, in contrast with previous observations (14).
ATP was shown previously to induce the processing and release of both IL-1 and IL-1 in macrophages (20). ATP induced the release of mature IL-1 from LPS-primed macrophages in this study, and release was inhibited by prior incubation of the cells with BAPTA-AM (Fig. 7). ATP and nigericin induced the release of IL-1 from WT, but not caspase-1 KO macrophages (Figs. 7 and 8). Thus, in response to ATP and nigericin, IL-1 and IL-1 release are caspase-1 dependent, although caspase-1 has no effect on the processing of pro- to mature IL-1 (34). It is possible that mature IL-1 influences the processing of IL-1. We have shown previously that in IL-1 KO microglia, ATP induces significantly lower levels of IL-1 release compared with WT cells (35). In addition, levels of IL-1 are significantly reduced in IL-1 KO mice (36). Another possibility for ATP-induced IL-1 release may be that a calpain is activated by caspase-1. Indeed, caspases are known to cleave the endogenous calpain inhibitor calpastatin, and thus promote calpain activation (37).
Ionomycin-induced IL-1 release still occurred from LPS-primed caspase-1 KO macrophages (Fig. 8B). This study thus illustrates two independent mechanisms for the release of mature IL-1. The first mechanism induced by ATP is dependent upon caspase-1/IL-1 and Ca2+ from ER stores, and the second dependent upon Ca2+ influx.
In conclusion, the data presented in this work indicate that in addition to K+ efflux (4, 14, 15), Ca2+ release from thapsigargin-sensitive Ca2+ stores is required for IL-1 processing and release from murine macrophages. In addition, Ca2+ entry across the plasmalemma is important for the processing and release of IL-1. Thus, Ca2+ from thapsigargin-sensitive stores and Ca2+ entry across the plasmalemma are important in regulating the differential secretion of IL-1 and IL-1.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nancy J. Rothwell, School of Biological Sciences, University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester M13 9PT, U.K. E-mail address: Nancy.rothwell{at}man.ac.uk
3 Abbreviations used in this paper: ER, endoplasmic reticulum; [Ca2+]i, intracellular Ca2+; KO, knockout; LDH, lactate dehydrogenase; WT, wild type.
Received for publication August 19, 2002. Accepted for publication January 10, 2003.
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 A. Wimmer, S. K. Khaldoyanidi, M. Judex, N. Serobyan, R. G. DiScipio, and I. U. Schraufstatter CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes Blood, December 1, 2006; 108(12): 3722 - 3729. [Abstract] [Full Text] [PDF]
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 V. H. J. Roberts, S. L. Greenwood, A. C. Elliott, C. P. Sibley, and L. H. Waters Purinergic receptors in human placenta: evidence for functionally active P2X4, P2X7, P2Y2, and P2Y6 Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2006; 290(5): R1374 - R1386. [Abstract] [Full Text] [PDF]
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 C. Yao, M. R. Karabasil, N. Purwanti, X. Li, T. Akamatsu, N. Kanamori, and K. Hosoi Tissue Kallikrein mK13 Is a Candidate Processing Enzyme for the Precursor of Interleukin-1beta in the Submandibular Gland of Mice J. Biol. Chem., March 24, 2006; 281(12): 7968 - 7976. [Abstract] [Full Text] [PDF]
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K. Okemoto, K. Kawasaki, K. Hanada, M. Miura, and M. Nishijima A Potent Adjuvant Monophosphoryl Lipid A Triggers Various Immune Responses, but Not Secretion of IL-1{beta} or Activation of Caspase-1 J. Immunol., January 15, 2006; 176(2): 1203 - 1208. [Abstract] [Full Text] [PDF]
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M. Hao, X. Li, M. A. Rizzo, J. V. Rocheleau, B. M. Dawant, and D. W. Piston Regulation of two insulin granule populations within the reserve pool by distinct calcium sources J. Cell Sci., December 15, 2005; 118(24): 5873 - 5884. [Abstract] [Full Text] [PDF]
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 N. Y. A. Hemdan, F. Emmrich, K. Adham, G. Wichmann, I. Lehmann, A. El-Massry, H. Ghoneim, J. Lehmann, and U. Sack Dose-Dependent Modulation of the In Vitro Cytokine Production of Human Immune Competent Cells by Lead Salts Toxicol. Sci., July 1, 2005; 86(1): 75 - 83. [Abstract] [Full Text] [PDF]
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F. Bianco, E. Pravettoni, A. Colombo, U. Schenk, T. Moller, M. Matteoli, and C. Verderio Astrocyte-Derived ATP Induces Vesicle Shedding and IL-1{beta} Release from Microglia J. Immunol., June 1, 2005; 174(11): 7268 - 7277. [Abstract] [Full Text] [PDF]
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L. C. Denlinger, G. Angelini, K. Schell, D. N. Green, A. G. Guadarrama, U. Prabhu, D. B. Coursin, P. J. Bertics, and K. Hogan Detection of Human P2X7 Nucleotide Receptor Polymorphisms by a Novel Monocyte Pore Assay Predictive of Alterations in Lipopolysaccharide-Induced Cytokine Production J. Immunol., April 1, 2005; 174(7): 4424 - 4431. [Abstract] [Full Text] [PDF]
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H. L. Wilson, S. E. Francis, S. K. Dower, and D. C. Crossman Secretion of Intracellular IL-1 Receptor Antagonist (Type 1) Is Dependent on P2X7 Receptor Activation J. Immunol., July 15, 2004; 173(2): 1202 - 1208. [Abstract] [Full Text] [PDF]
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 C. Andrei, P. Margiocco, A. Poggi, L. V. Lotti, M. R. Torrisi, and A. Rubartelli From The Cover: Phospholipases C and A2 control lysosome-mediated IL-1{beta} secretion: Implications for inflammatory processes PNAS, June 29, 2004; 101(26): 9745 - 9750. [Abstract] [Full Text] [PDF]
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R. Sluyter, A. N. Shemon, and J. S. Wiley Glu496 to Ala Polymorphism in the P2X7 Receptor Impairs ATP-Induced IL-1{beta} Release from Human Monocytes J. Immunol., March 15, 2004; 172(6): 3399 - 3405. [Abstract] [Full Text] [PDF]
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