title: 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 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 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.
authors.affiliation: Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Ferrara, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Gulinelli
authors.forename: Erica
authors.initials: E
authors.lastname: Salaro
authors.forename: Marta
authors.initials: M
authors.lastname: Vuerich
authors.forename: Dania
authors.initials: D
authors.lastname: Bozzato
authors.forename: Cinzia
authors.initials: C
authors.lastname: Pizzirani
authors.forename: Giorgio
authors.initials: G
authors.lastname: Bolognesi
authors.forename: Marco
authors.initials: M
authors.lastname: Idzko
authors.forename: Francesco
authors.initials: F
authors.lastname: Di Virgilio
authors.forename: Davide
authors.initials: D
authors.lastname: Ferrari
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000081:Acetamides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D055252:Cell-Derived Microparticles / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D020382:Interleukin-18 / Q000276:immunology* / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D011112:Polymyxin B / Q000494:pharmacology; D011498:Protein Precursors / Q000276:immunology* / Q000378:metabolism; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D058848:Receptors, Purinergic P2X4 / Q000276:immunology* / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000276:immunology* / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013777:Tetrazoles / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000276:immunology* / Q000378:metabolism
chemical_list: C515928:(N-(1-(((cyanoimino)(5-quinolinylamino) methyl) amino)-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide); C523668:3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methylpyridine; D000081:Acetamides; D000900:Anti-Bacterial Agents; D061207:Calcium Ionophores; D020382:Interleukin-18; D008070:Lipopolysaccharides; D011498:Protein Precursors; D058911:Purinergic P2X Receptor Agonists; D058920:Purinergic P2X Receptor Antagonists; D011725:Pyridines; D011804:Quinolines; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; D013449:Sulfonamides; C495345:TLR4 protein, human; D013777:Tetrazoles; D051197:Toll-Like Receptor 4; C507035:ethyl 6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate; D000001:Calcimycin; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D011112:Polymyxin B