Extracellular ATP, released upon microbial infection, cell damage or inflammation, acts as an alert signal towards immune cells by activating P2 receptors. The nucleotide causes microvesicle shedding from immune and nonimmune cells. Here we show that IL-18 associates with microvesicles (MV) shed by human ex vivo macrophages upon P2X receptor stimulation. MV shedding was potently induced by ATP and by the P2X7 agonist BzATP, while it was greatly reduced by P2X irreversible inhibitor-oxidized ATP and by the specific P2X7 inhibitors KN-62, A-740003 and A-438079. Peculiarly, the P2X7 subtype was highly present in the MV, while on the contrary the P2X3 and P2X4 subtypes were almost absent. The Ca2+ ionophore A23187 mimicked the effect of BzATP suggesting that an intracellular Ca2+ 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 pre-treatment of cells with lipopolysaccharide, as the pro-cytokine was already present in unprimed macrophages and did not decrease by incubating cells with the LPS-binding antibiotic polymyxin B nor by 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.
IL-18 associates to microvesicles shed from human macrophages by a LPS/TLR-4 independent mechanism in response to P2X receptor stimulation
GULINELLI, Sara;SALARO, ERICA;VUERICH, Marta;PIZZIRANI, Cinzia;BOLOGNESI, Giorgio;DI VIRGILIO, Francesco;FERRARI, Davide
2012
Abstract
Extracellular ATP, released upon microbial infection, cell damage or inflammation, acts as an alert signal towards immune cells by activating P2 receptors. The nucleotide causes microvesicle shedding from immune and nonimmune cells. Here we show that IL-18 associates with microvesicles (MV) shed by human ex vivo macrophages upon P2X receptor stimulation. MV shedding was potently induced by ATP and by the P2X7 agonist BzATP, while it was greatly reduced by P2X irreversible inhibitor-oxidized ATP and by the specific P2X7 inhibitors KN-62, A-740003 and A-438079. Peculiarly, the P2X7 subtype was highly present in the MV, while on the contrary the P2X3 and P2X4 subtypes were almost absent. The Ca2+ ionophore A23187 mimicked the effect of BzATP suggesting that an intracellular Ca2+ 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 pre-treatment of cells with lipopolysaccharide, as the pro-cytokine was already present in unprimed macrophages and did not decrease by incubating cells with the LPS-binding antibiotic polymyxin B nor by 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.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
