Silica and Asbestos Induce IL-1β Secretion in a Nalp3-Dependent Manner.

Nalp3 along with the adaptor molecule ASC can activate caspase-1 in response to a variety of stimuli, including ATP, nigericin, maitotoxin, MSU, and the pathogens Listeria monocytogenes and Staphylococcus aureus (12–14). We used macrophages deficient in specific components of the Nalp3 inflammasome to test whether they were required for silica-induced IL-1β secretion. LPS-primed Nalp3-, ASC- and caspase-1-deficient macrophages all displayed a marked defect in their ability to secrete IL-1β in response to silica compared with macrophages from WT mice (Fig. 2A). In contrast, macrophages deficient in the NLR molecule IPAF, which is important for macrophage caspase-1 activation in response to infection with type III and type IV secretion system carrying Gram-negative bacteria (16–18), had an intact response to silica and were capable of secreting IL-1β (Fig. 2A). In addition, LPS-primed Nalp3-, ASC-, and caspase-1-deficient macrophages were also unable to secrete the other caspase-1-processed cytokine IL-18 in response to silica (Fig. 2B).

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Fig. 2.

Silica- and asbestos-induced IL-1β secretion is dependent on Nalp3. LPS-primed macrophages from WT, caspase-1-, ASC-, Nalp3-, or IPAF-deficient mice were stimulated with 50 μg/cm² silica. Culture supernatants were collected at 4 h or at the indicated time after stimulation (A–C) and IL-1β and IL-18 release into culture supernatants was measured by ELISA (A and B). Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of three (A) and two (B) separate experiments. (C) Lysates from LPS-primed WT, ASC-, NALP3-, or IPAF-deficient macrophages stimulated with 50 μg/cm² silica for the indicated times were immunoblotted with antibodies against the p10 subunit of caspase-1. (D) LPS-primed macrophages from WT and NALP3-deficient mice were stimulated with either 40 μg/cm² asbestos or 50 μg/cm² titanium dioxide. Culture supernatants were collected 6 h later and IL-1β release into culture supernatants was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of three separate experiments. *, Nondetectable.

Caspase-1 activation involves autocatalytic processing of the 45-kDa pro-caspase-1 to generate two subunits, p20 and p10. Caspase-1 activation in silica-stimulated LPS-primed WT macrophages was detected by Western blotting by the appearance of the p10 cleavage product 2 h after stimulation with silica (Fig. 2C). We did not observe caspase-1 activation in response to silica in either Nalp3- or ASC-deficient LPS-primed macrophages (Fig. 2C). As expected, IPAF-deficient LPS-primed macrophages did not display any defect in caspase-1 activation in response to silica (Fig. 2C). These data identify the Nalp3 inflammasome to be required for caspase-1-mediated IL-1β and IL-18 secretion in response to stimulation with silica.

The inhalation of asbestos leads to a similar fibrotic syndrome, asbestosis, characterized by chronic inflammation and insidious deterioration of lung function, which usually first manifests more than 15 years after the initial exposure to asbestos. Asbestos, but not titanium dioxide, stimulation of LPS-primed WT macrophages resulted in the secretion of IL-1β in a Nalp3-dependent manner (Fig. 2D). This suggests a common mechanism for inflammation in response to the pro-fibrotic agents silica and asbestos requiring Nalp3, which is not shared by other, nonfibrotic, inhalant particles.

Silica-Induced Cytotoxicity Is Independent of Nalp3.

It has been shown that treatment of macrophages with silica is cytotoxic (19). To determine whether silica-induced cytotoxicity was associated with Nalp3, we assessed the degree of cell death in WT and Nalp3-deficient macrophages after silica treatment. We confirmed the cytotoxic effect of silica and found it to be equivalent in macrophages from WT and Nalp3-deficient mice (Fig. 3A). These data demonstrate that silica-induced cytotoxicity is not dependent on Nalp3, and that IL-1β release was not a consequence of cell lysis, because WT and Nalp3-deficient macrophages had similar amounts of cell death but only the former released IL-1β.

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Fig. 3.

Silica-induced IL-1β secretion requires internalization of silica, a potassium efflux, and generation of reactive oxygen species. (A) LPS-primed WT macrophages were stimulated with the indicated amount of silica. Culture supernatants were collected 4 h later and cytotoxicity was measured by lactate dehydrogenase (LDH) release and expressed as a percentage of LDH release by Triton X-100 detergent. (B) LPS-primed WT macrophages were incubated with 10 μM cytochalasin B or 20 μM cytochalasin D for 10 min before the addition of 50 μg/cm² silica or 5 mM ATP. (C) LPS-primed macrophages were incubated in either high Na⁺- or high K⁺-containing medium and then stimulated with either 50 μg/cm² silica or 5 mM ATP. ATP-treated cells had their medium replaced with fresh medium after 20 min. Four hours after initial stimulation, culture supernatants were collected and IL-1β release was measured by ELISA. (A–C) Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of two separate experiments. (D and E) WT and Nalp3−/− macrophages were stimulated with the indicated amount of silica, and ROS production was measured by luminol-amplified chemiluminescence. RLU, relative light units. (F and G) LPS-primed WT macrophages were incubated with 10 μM or the indicated amount of diphenyleneiodonium chloride (DPI) for 10 min before the addition of 50 μg/cm² silica. Four hours after initial stimulation, culture supernatants were collected and IL-1β release was measured by ELISA (F). Determinations were performed in triplicate and expressed as the mean ± SEM and are representative of three separate experiments. At the indicated time after stimulation lysates were collected and immunoblotted with antibodies against the p10 subunit of caspase-1, caspase-3, and actin (G). *, Nondetectable.

Silica-Induced IL-1β Secretion Requires Crystal Internalization and Potassium Efflux.

To understand how silica might activate the Nalp3 inflammasome, we tested whether the endocytic ability of macrophages was required for silica-stimulated IL-1β production. Inhibiting actin polymerization with either cytochalasin B or cytochalasin D inhibited IL-1β production by silica in LPS-primed macrophages (Fig. 3B). Neither cytochalasin B nor cytochalasin D reduced IL-1β production in response to stimulation with ATP, which uses the P2X7 receptor to activate the Nalp3 inflammasome, confirming that macrophages were still viable and capable of secreting Nalp3-dependent IL-1β (Fig. 3B). These data suggest that, unlike ATP, endocytosis of silica by macrophages is needed to activate the Nalp3 inflammasome in response to silica for the resultant processing and secretion of proinflammatory cytokines. Endocytosis of particulates may be occurring through the scavenger receptors SR-A and MARCO, which have been implicated in playing an important role in the inflammatory response to silica (20). Further studies will be required to determine whether these receptors are also required for silica-induced Nalp3 inflammasome activation.

A number of diverse stimuli can activate the Nalp3 inflammasome. However, a common step shared by these stimuli to induce Nalp3-dependent caspase-1 activation is the efflux of cellular potassium (21). Preventing the potassium efflux by increasing extracellular potassium inhibits caspase-1 activation in response to stimuli that activate Nalp3 but not stimuli that activate caspase-1 through IPAF (21). Increased extracellular potassium significantly inhibited silica-induced IL-1β production from macrophages (Fig. 3C). As expected, increased extracellular potassium also inhibited ATP-mediated IL-1β production, which is also dependent on Nalp3. Silica therefore is a previously unrecognized Nalp3 stimulus, which like other Nalp3 stimuli requires a cellular potassium efflux to activate caspase-1.

Silica-Induced ROS Are Required for Nalp3 Inflammasome Activation.

There are two main sources of cellular ROS. The first is the phagocyte NADPH oxidase, which generates superoxide anions as part of the antimicrobial response to ingested microbes (22). In the resting phagocyte, components of the NADPH oxidase are spatially segregated, with flavocytochrome b₅₅₈ in the membrane and p47^phox, p67^phox, and Rac in the cytoplasm. Upon stimulation, the cytosolic components translocate to the membrane and assemble into a functioning enzyme complex, shuttling electrons from cytoplasmic NADPH to molecular oxygen to generate extracellular or phagosomal superoxide anion. Mitochondria have also long been known to generate reactive oxidants and are a major source of cellular ROS (22). The mitochondrial respiratory chain is made up of four electron-transporting complexes and a proton-translocating complex, with the majority of intracellular ROS produced during mitochondrial respiration by complex I. ROS have also been shown to play important signaling functions within the cell either for homeostatic functions or to signal stress. This is particularly the case for the mitochondria, which can either confine its production of ROS locally or flood the cell with oxidants, causing damage to itself and the cell in the process.

The Nalp3 inflammasome activator ATP has been shown to be capable of inducing the production of ROS, which are important for caspase-1 activation (23). As silica can also induce ROS production from macrophages (19) we examined whether this was required for caspase-1 mediated IL-1β secretion. Using HRP-dependent luminol-enhanced chemiluminescence to detect both intra- and extracellular ROS, we confirmed that silica induced ROS production from macrophages (Fig. 3D) and that the response was blocked by preincubation of macrophages with the inhibitor of NADPH oxidase diphenyleneiodonium chloride (DPI) (data not shown). DPI also markedly inhibited silica-induced IL-1β secretion and caspase-1 activation (Fig. 3 F and G), thereby suggesting that ROS signaling was required for silica-induced Nalp3 inflammasome activation. DPI did not inhibit silica-induced caspase-3 activation in LPS-primed macrophages (Fig. 3G). Nalp3-deficient macrophages were capable of producing ROS in response to silica (Fig. 3E), indicating that ROS signaling is likely occurring upstream of Nalp3. Although these data demonstrate a role for ROS in silica-induced IL-1β secretion, they do not distinguish between the two most likely sources for ROS in stimulated cells, because DPI can also exert inhibitory effects on mitochondrial ROS production in addition to NADPH oxidase. However, taken together these studies clearly define a novel signaling pathway used to activate the Nalp3 inflammasome.

Macrophages.

Thioglycollate-elicited peritoneal macrophages were generated by injecting 1 ml of 3% thioglycollate solution into the peritoneal cavity of mice. Three to 4 days later macrophages were collected by peritoneal lavage and plated in DMEM supplemented with 10% FCS, 2 mM l-glutamine, 100 units/ml penicillin G, and 100 μg/ml streptomycin. Unless indicated, macrophages were activated by stimulating with 50 ng/ml LPS from Escherichia coli serotype O111:B4 (InvivoGen) for 4–12 h before stimulation with silica or asbestos.

Human alveolar macrophages were obtained from normal volunteers by bronchoalveolar lavage as described in ref. 29, and the procedure was approved by the Committee for Investigations Involving Human Subjects at the University of Iowa.

In Vitro Stimulation of Macrophages.

Macrophages that were either unstimulated or had been activated with 50 ng/ml LPS for 4–12 h were stimulated with either 50 μg/cm² silica (Min-U-Sil-5; Pennsylvania Glass Sand), 40 μg/cm² crocidolite asbestos (North American Insulation Manufacturers Association Fiber Repository), or 50 μg/cm² titanium dioxide (Sigma) unless otherwise indicated. Four hours, or at the indicated time, after stimulation supernatants were collected. Culture supernatants were then assayed for IL-1β and TNFα and IL-6. Antibody pairs for the IL-1β ELISAs were from R&D Systems. Antibody pairs for the TNFα and IL-6 ELISAs were from eBiosciences. Macrophage cell death was determined 4 h after stimulation with silica by using a cytotoxicity (LDH release) detection kit (Promega). The inhibitors cytochalasin B (10 μM), cytochalasin D (20 μM), and diphenyleneiodonium chloride (DPI) (2 μM and 10 μM) were added 10 min before the addition of silica.

RT-PCR.

RNA was isolated from macrophages by using the Qiagen RNeasy Mini kit and following the manufacturer's instructions. cDNA was synthesized by using SuperScript II reverse transcriptase (Invitrogen). The primers used for RT-PCR amplification were as follows: HPRT, 5′-GTTGGATACAGGCCAGACTTTGTT and 3′-GAGGGTAGGCTGGCCTATAGGCT; IL-12p40, 5′-ATGGCCATGTGGGAGCTGGAGAAAG and 3′-GTGGAGCAGCAGATGTGAGTGGCT; IL-1β, 5′-GACAGTGATGAGAATGACCTGTTC and 3′-CCTGACCACTGTTGTTTCCC.

Inhibition of Potassium Efflux.

To inhibit potassium efflux, macrophages were primed with 50 ng/ml LPS for 12 h and then medium was replaced with serum-free buffer with 150 mM KCl (10 mM Hepes, 5 mM NaH₂PO₄, 150 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 1% BSA, pH 7.4). In parallel, a buffer with 150 mM NaCl was used (10 mM Hepes, 150 mM NaCl, 5 mM KH₂PO₄, 1 mM MgCl₂, 1 mM CaCl₂, 1% BSA, pH 7.4).

ROS Measurement.

Production of ROS was measured by the peroxidase-dependent luminol-amplified chemiluminescence technique, as previously described (30). Briefly, chemiluminescence was monitored with a Lumistar Galaxy (BMG Labtech) luminometer. Macrophages were plated on white polystyrene microtiter plates (Optiplate, Perkin–Elmer) in Hanks balanced salt solution containing 1 mM CaCl₂, 1.5 mM MgCl₂, 10 mM glucose, and 1% human serum albumin. Cells were incubated at 37°C in the presence of 50 μM luminol and 4 units of horseradish peroxidase. Stimuli were added to the respective wells and chemiluminescence was followed continuously for 60 min. Assays were run in duplicate.

Western Blotting.

Electrophoresis of proteins was performed by using the NuPAGE system (Invitrogen) according to the manufacturer's protocol. Briefly, after stimulation with silica, macrophages and supernatants were lysed in lysis buffer [50 mM Tris·HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, and a protease inhibitor mixture (Roche)] and stored at −80°C until analyzed. Proteins were separated on a NuPAGE gel and transferred to a PVDF membrane by electroblotting. To detect caspase-1 a rabbit polyclonal anti-mouse casapse-1 p10 antibody (Santa Cruz Biotechnology) was used. Caspase-3 and actin were detected by using antibodies from Cell Signaling and Calbiochem, respectively.

We thank David Meyerholz for pathology analysis. We thank William Nauseef and Joao Pedra for critical review of the manuscript and Anthony Coyle, Ethan Grant, and John Bertin for providing mutant mice. This work was supported by grants from the Ellison Foundation (R.A.F.), National Institutes of Health Grant K08 AI065517 (F.S.S.), National Institutes of Health Grant K08 AI067736 (S.L.C.), National Institutes of Health Grant ES014871 (A.B.C.), and National Institutes of Health Grant ES015981 (A.B.C.). R.A.F. is an investigator of the Howard Hughes Medical Institute.