IL‐18 associates to microvesicles shed from human macrophages by a LPS/TLR‐4 independent mechanism in response to P2X receptor stimulation

Abstract

Extracellular ATP, released upon microbial infection, cell damage, or inflammation, acts as an alert signal toward immune cells by activating P2 receptors. The nucleotide causes microvesicle (MV) shedding from immune and nonimmune cells. Here, we show that IL-18 associates with MVs shed by human ex vivo macrophages upon P2X receptor stimulation.MV shedding was potently induced by ATP and by the P2X7 agonist 3 -benzoylbenzoyl adenosine 5 -triphosphate, while it was greatly reduced by P2X irreversible inhibitoroxidized ATP and by the specific P2X7 inhibitors KN-62, A-740003, and A-438079. Peculiarly, the P2X7 subtype was highly present in the MVs, while on the contrary the P2X3 and P2X4 subtypes were almost absent. The Ca 2+ ionophore A23187 mimicked the effect of 3 -benzoylbenzoyl adenosine 5 -triphosphate suggesting that an intracellular Ca 2+ increase was sufficient to evoke MV shedding. Caspase inhibitors Ac-YVAD-CMK or Z-YVAD-CMK did not block the cleavage of MV-associated pro-IL-18. Pro-IL-18 formation in macrophages did not require pretreatment of cells with LPS, as the procytokine was already present in unprimed macrophages and did not decrease by incubating cells with the LPS-binding antibiotic polymyxin B nor with the TLR-4 intracellular inhibitor CLI-095. These data reveal a nucleotide-based mechanism responsible for the shedding of MV to which IL-18 is associated.Keywords: Extracellular ATP r Human macrophages r IL-18 r P2 receptors r P2X7 Supporting Information available online IntroductionMicrovesicles (MVs) are small vesicles derived from the plasma membrane of eukaryotic cells [1,2]. MVs are gaining momenCorrespondence: Dr. Davide Ferrari e-mail: dfr@unife.it tum as they have been isolated in tumors, infectious and autoimmune diseases, and a role in delivery different messages to the cells has also been demonstrated [2][3][4]. They can transport and deliver proteins, enzymes, mRNA, or microRNA. Recent * These authors contributed equally to this work.C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2012. 42: 3334-3345 Innate immunity 3335 observations have shown that MVs shed by mesenchymal stem cells play a role in inducing peripheral tolerance and modulation of the immune response [5]. Proinflammatory cytokines such as IL-1β and IL-18 are leaderless secreted proteins. It has been shown that IL-1β is efficiently released from immune cells in association to MVs containing the cytokine. Shedding of MV requires stimulation of membrane receptors for the extracellular nucleotides [6]. IL-18, previously named IFN-γ-inducing factor for its ability to stimulate IFN-γ production in T lymphocytes and natural killer cells, is one of the main cytokines contributing to the pathogenesis of autoimmune and inflammatory diseases [7]. Besides lymphocytes, IL-18 is secreted by a variety of cell types including epithelial cells, keratinocytes, synovial fibroblasts, monocytes, macrophages, and DCs [8,9], thus inducing the synthesis of other proinflammatory cytokines (IFN-γ, IL-1β...

Alveolar macrophage-derived microvesicles mediate acute lung injury

“…They were either primed for 1 hour with 1 µg/mL of LPS (37°C) to attain a proinflammatory phenotype24 or incubated with phosphate-buffered saline (PBS) alone, and then stimulated with 6 mM ATP,25 1 mM ecto-ATPase inhibitor (ARL67156) and 40 µM calcium ionophore (A23187)26 for 2 hours to generate MVs of either ‘inflammatory’ or ‘non-inflammatory’ phenotype in vitro. MVs and MV-depleted supernatants were separated by centrifugation (see online supplementary information) and incubated with MLE-12 cells for 4 hours, with and without 10 µg/mL polyclonal TNF-neutralising antibody.…”

Type I IFNs and IL-18 Regulate the Antiviral Response of Primary Human γδ T Cells against Dendritic Cells Infected with Dengue Virus

“…The reagents used and their final concentration in culture are as follows: 5 mg/ml B18R recombinant protein (eBioscience), 10 mg/ml anti-IL-18 (clone 125-2H; R&D Systems), 10 mg/ml anti-TNF-a (clone MAb1; eBioscience), 10 mg/ml IL-1R antagonist (R&D Systems), 10 mg/ml anti-IL-7 (clone BVD10-40F6; BioLegend), 10 mg/ml anti-IL-12 (clone C8.6; eBioscience), 10 mg/ml anti-IL-15 (clone MAB2471, R&D Systems), and 10 mg/ml mouse IgG1 isotype control (clone MOPC-21; eBioscience). 3-[[5-(2,3-Dichlorophenyl)-1H-tetrazol-1-yl]methyl]pyridine hydrochloride (15D; Tocris) was used at a final concentration of 100 mM as a specific antagonist of the P2X 7 receptor (24). DV-infected DC were treated with mevastatin (Sigma-Aldrich) at a final concentration of 5 mM for 16 h to block the mevalonate pathway of isopentenyl pyrophosphate (IPP) synthesis (25); they were subsequently cocultured with PBMC for an additional 4 h in the presence of mevastatin.…”

Extracellular ATP induces the rapid release of HIV-1 from virus containing compartments of human macrophages

“…7A, Left). Because in addition to P2X7R, oATP has been reported to bind to P2X1R and P2X2R, we stimulated infected MDM with eATP after their short preincubation with A-438079, a selective inhibitor of P2X7R (43,44), which prevented the eATP-dependent virion release from MDM (Fig. 7B, Left).…”