Key points are not available for this paper at this time.
Interleukin (IL)-1β and IL-18 are structurally similar proteins that require caspase-1 processing for activation. Both proteins are released from the cytosol by unknown pathway(s). To better characterize the release pathway(s) for IL-1β and IL-18 we evaluated the role of lipopolysaccharide priming, of interleukin-1β-converting enzyme (ICE) inhibition, of human purinergic receptor (P2X7) function, and of signaling pathways in human monocytes induced by ATP. Monocytes rapidly processed and released both IL-1β and IL-18 after exogenous ATP. Despite its constitutive cytosolic presence, IL-18 required lipopolysaccharide priming for the ATP-induced release. Neither IL-1β nor IL-18 release was prevented by ICE inhibition, and IL-18 release was not induced by ICE activation itself. Release of both cytokines was blocked completely by a P2X7 receptor antagonist, oxidized ATP, and partially by an antibody to P2X7 receptor. In evaluating the signaling components involved in the ATP effect, we identified that the protein-tyrosine kinase inhibitor, AG126, produced a profound inhibition of both ICE activation as well as release of IL-1β/IL-18. Taken together, these results suggest that, although synthesis of IL-1β and IL-18 differ, ATP-mediated release of both cytokines requires a priming step but not proteolytically functional caspase-1. Interleukin (IL)-1β and IL-18 are structurally similar proteins that require caspase-1 processing for activation. Both proteins are released from the cytosol by unknown pathway(s). To better characterize the release pathway(s) for IL-1β and IL-18 we evaluated the role of lipopolysaccharide priming, of interleukin-1β-converting enzyme (ICE) inhibition, of human purinergic receptor (P2X7) function, and of signaling pathways in human monocytes induced by ATP. Monocytes rapidly processed and released both IL-1β and IL-18 after exogenous ATP. Despite its constitutive cytosolic presence, IL-18 required lipopolysaccharide priming for the ATP-induced release. Neither IL-1β nor IL-18 release was prevented by ICE inhibition, and IL-18 release was not induced by ICE activation itself. Release of both cytokines was blocked completely by a P2X7 receptor antagonist, oxidized ATP, and partially by an antibody to P2X7 receptor. In evaluating the signaling components involved in the ATP effect, we identified that the protein-tyrosine kinase inhibitor, AG126, produced a profound inhibition of both ICE activation as well as release of IL-1β/IL-18. Taken together, these results suggest that, although synthesis of IL-1β and IL-18 differ, ATP-mediated release of both cytokines requires a priming step but not proteolytically functional caspase-1. interleukin interleukin-1β-converting enzyme protein-tyrosine kinase human purinergic receptor mitogen-activated protein kinase/extracellular signal-regulated kinase kinase protein kinases A and C, respectively enzyme-linked immunosorbent assay acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone extracellular signal-regulated kinase lipopolysaccharide polyacrylamide gel electrophoresis IL-1β1 and IL-18 are proinflammatory cytokines that require processing by the IL-1-converting enzyme, caspase-1, at specific aspartic acid residues to generate functional molecules (1Howard A.D. Kostura M.J. Thornberry N. Ding G.J.F. Limjuco G. Weidner J. Salley J.P. Hogquist K.A. Chaplin D.D. Mumford R.A. Schmidt J.A. Tocci M.J. J. Immunol. 1991; 147: 2964-2969PubMed Google Scholar, 2Ghayur T. Banerjee S. Hugunin M. 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P2X7 is a 595-amino acid polypeptide with two membrane-spanning domains and intracellular N- and C-terminal domains (22Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. Science. 1996; 272: 735-738Crossref PubMed Scopus (1536) Google Scholar, 23Rassendren F. Buell G.N. Virginio C. Collo G. North R.A. Surprenant A. J. Biol. Chem. 1997; 272: 5482-5486Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). P2X7receptor is the only pore-forming P2X family member, and its activation results in the opening of a cationic channel with increased permeability to calcium and intracellular depolarization (20Dubyak G.R. El-Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar). The present work focuses on release mechanisms by taking advantage of the dramatic ability of ATP to telescope the processing and release down to a 15–30-min interval while enhancing the overall release signal (15Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Melchiorri L. Baricordi O.R. Di Virgilio F. J. Immunol. 1997; 159: 1451-1458PubMed Google Scholar). In this report we examine the mechanism of ATP-induced release of IL-18 and compare it to IL-1β in human monocytes. We show that fresh monocytes express functional P2X7 receptors that modulate IL-1β and IL-18 processing and release in response to ATP. Of note, our data suggest that ATP stimulation alone is insufficient to trigger IL-18 processing and release, since priming with LPS was shown to be necessary. Additionally, the ATP effect on release does not require functional caspase-1, confirming for IL-18 what has been previously shown for IL-1β, i.e. precursor cleavage is not required for release. Finally, attempts to characterize the signaling pathway involved in the ATP effect show that a protein-tyrosine kinase inhibitor, AG126, can potently block activation of caspase-1 and IL-1β/IL-18 release events, whereas mitogen-activated protein kinase, PKC and PKA inhibitors do not. ProIL-1β-specific rabbit polyclonal antibody (amino acids, 3–21) was developed in our laboratory. Human recombinant IL-1β, biotinylated IL-18, anti-human monoclonal IL-1β antibody (clone 8516), and IL-18 ELISA kit were purchased from R Google Scholar). monocytes. monocytes were at in with at in cells were for in the of LPS and in the of caspase-1 (ICE) human monocytes were with ATP and for an was from the cells and at for and the and were were in and and The and were by at for the of in of and were in The protein in was the protein assay The proteins were by and to on were blocked with in for at with the was blocked with in The were with as by and protein were by monocytes were in alone in LPS for in LPS cells were with monoclonal antibody to P2X7 as G. A.D. Collo G. M. S. D. C. R. 1998; PubMed Google Scholar). cells were with in and in monoclonal P2X7 an antibody for at by cells were for the of P2X7 receptor monocytes were for was were developed in our to and IL-1β as M.D. Dare H.A. Winnard A.V. Parker J.M. Miller D.K. J. Immunol. 1997; 159: 5964-5972PubMed Google Scholar). The antibody has been since the anti-human monoclonal IL-1β antibody (clone R Full Text PDF PubMed Scopus Google Scholar). To the for caspase-1 in ATP-induced IL-1β and IL-18 release, we caspase-1 with the We IL-1β and IL-18 release with to both cytokines in the precursor M.D. Dare H.A. Winnard A.V. Parker J.M. Miller D.K. J. Immunol. 1997; 159: 5964-5972PubMed Google Scholar). Human monocytes were with and LPS in we and previously shown M.D. Dare H.A. Winnard A.V. Parker J.M. Miller D.K. J. Immunol. 1997; 159: 5964-5972PubMed Google Scholar, J.M. Di Virgilio F. J. Immunol. PubMed Scopus Google Scholar), the of an ICE prevented IL-1β release from monocytes not a significant of was in a ELISA and was shown for IL-1β, after LPS priming, ATP induced monocytes to release IL-18 and Although ATP alone not the release of IL-18 from cells with caspase-1 inhibitor, ICE inhibition not IL-18 release from monocytes data suggest that release of IL-1β and IL-18 does not require as has been shown for IL-1β, the processing of IL-18 is not required for its To that the ATP not only release but processing of IL-1β and IL-18, were by and We the of ATP on the processing of IL-18 and IL-1β in monocytes in monocytes with LPS alone LPS and the precursor of IL-18 and the of IL-1β were in In of IL-1β was of the precursor of IL-18 were In a of IL-18 was In the the of was not but a of IL-1β was In the of only precursor of IL-18 and IL-1β were in of monocytes A and was in the of IL-18 and IL-1β in the of show that of and IL-18 but does not block the release. both IL-18 and IL-1β require functional caspase-1 for processing has been that ATP caspase-1 in human monocytes J. Gabel C.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is not the to ATP in our ICE in human monocytes. we the effect of ATP and on activation of ICE by Monocytes were in the of LPS and with without for ATP was and cells were for an In and the precursor and of ICE were but of was However, monocytes with ATP a level of and of was blocked by the of in the of LPS priming, ATP alone effect on IL-18 processing and release, it was important to ATP alone shown in ATP without LPS activated ICE to the in both and monocytes. the data from ELISA and with the of ICE show that ATP not only ICE but the processing and release of IL-1β and Although the activation of ICE and the of these precursor it to the release of and in the of LPS priming, ATP-induced ICE activation does not processing and release. although ATP ICE it requires an signal to processing and release of IL-1β and A of to suggest that ATP-induced release of IL-1β in monocytes occurs via P2X7 receptors (15Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Melchiorri L. Baricordi O.R. Di Virgilio F. J. Immunol. 1997; 159: 1451-1458PubMed Google Scholar, G. A.D. Collo G. M. S. D. C. R. 1998; PubMed Google Gabel C.A. J. Immunol. 1998; Google Scholar). Human monocytes for in the of LPS monocytes in the of P2X7 receptors G. A.D. Collo G. M. S. D. C. R. 1998; PubMed Google Scholar). However, it is not fresh human monocytes for with without LPS express P2X7 To this we human monocytes in the of LPS for for the of P2X7 receptors by and a significant signal for P2X7 receptor was in human monocytes with the the with P2X7 antibody was only in cells in the of LPS for the results of A was in and that was with the data that fresh monocytes express P2X7 receptors that are only increased by of P2X7 receptor on human monocytes. from monocytes were to to and with polyclonal receptor antibody as with P2X7 receptor with of and P2X7 was as human We the monocytes express functional P2X7 we to the P2X7 receptor antagonist, oxidized ATP, and a antibody the release of IL-1β in human monocytes. with oxidized ATP for completely the ATP-induced release of IL-1β in human monocytes with monoclonal antibody blocked the release of IL-1β but to a The data suggest that the P2X7 receptor is for the ATP We that protein kinases a significant role in the release of IL-18 and IL-1β from human monocytes. we the of inhibitors of protein-tyrosine protein kinase C, and protein kinase A on release of IL-1β from human monocytes and receptor kinase inhibitors and PKA PKC pathway inhibitors effect, AG126, a inhibitor, prevented ATP-induced release of IL-1β from monocytes The effect was and not to the cells in the in the that IL-1β release is by an of protein kinase inhibitors on ATP release of IL-1β from of were with LPS for with inhibitors as for and with ATP for IL-1β was by and data are as inhibition to LPS were with LPS for with inhibitors as for and with ATP for IL-1β was by and data are as inhibition to LPS in a is a of and and activation while from J.A. P. 1996; PubMed Scopus Google Scholar, A. A. M. A. N. A. Science. 1994; PubMed Scopus Google Scholar). we the effect on IL-1β and IL-18 release is to the block in inhibition of activation. ELISA we the of inhibitors A and AG126, mitogen-activated protein kinase kinase inhibitors and and on ATP-induced release of IL-1β in monocytes IL-1β release in a whereas the A and the inhibitors and not ATP induced IL-1β release To examine the effect was to the inhibition of we ATP-induced IL-1β release in the of a of the inhibition of release, IL-1β release by Of note, the activated release of IL-1β We evaluated ATP-induced the classic and prevented not. In the inhibitors not IL-1β and IL-18 release, but the results that the activation of mitogen-activated protein kinase pathway is not for ATP-induced IL-1β release from monocytes. is that the kinase by caspase-1 activation. We the inhibition of release of cytokines by is by the of To this we the ATP-induced processing and release of IL-1β and IL-18 in the of kinase of the for IL-1β and IL-18 the ELISA it for IL-1β, the processing and release of The A and the not ATP-induced processing and release of the Finally, we the effect of on caspase-1 activation by the ICE as the of ICE was not and inhibitors effect on ATP-induced activation of ICE the inhibition of IL-1β release by with the inhibition of ATP-induced ICE and the processing of ICE to be regulated by protein-tyrosine The processing of precursor IL-1β and IL-18 and the are in Both and are proteins that require cleavage by caspase-1 for activation (8Dinarello C.A. Ann. N. Y. Acad. Sci. 1998; 856: 1-11Crossref PubMed Scopus (430) Google Scholar, A.J. Fantuzzi G. Dinarello C.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2256-2261Crossref PubMed Scopus (332) Google Scholar, C.A. 1996; PubMed Google Scholar). Both IL-1β and IL-18 share significant structural homology (4Bazan J.F. Timans J.C. Kastelein R.A. Nature. 1996; 379 (591): 591Crossref PubMed Scopus (265) Google Scholar, C.A. J. Allergy Clin. Immunol. 1999; 103: 11-24Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar), and both a sequence required for although the release of IL-1β and IL-18 to be to the processing of precursor by caspase-1, functional ICE has not been in macrophages, monocytes the processing and release of IL-1β was induced by LPS M.D. Dare H.A. Winnard A.V. Parker J.M. Miller D.K. J. Immunol. 1997; 159: 5964-5972PubMed Google Scholar, J.M. J. Kostura M.J. Miller D.K. J. Immunol. 1994; Google Scholar). studies that extracellular ATP both the and the of the processing and release of from and human and cells with LPS and monocytes (15Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Melchiorri L. Baricordi O.R. Di Virgilio F. J. Immunol. 1997; 159: 1451-1458PubMed Google R.J. Stam E.J. Downs J.T. Otterness I.G. J. Immunol. 1995; 154: 2821-2828PubMed Google Scholar, J.M. Di Virgilio F. J. Immunol. PubMed Scopus Google Scholar, D. Chiozzi P. Falzoni S. Dal Susino M. Collo G. Buell G. Di Virgilio F. 1997; PubMed Scopus Google Scholar, D. Chiozzi P. Falzoni S. S. Di Virgilio F. J. 1997; PubMed Scopus Google Scholar). release of IL-1β occurs via activation of the P2X7 receptor (15Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Melchiorri L. Baricordi O.R. Di Virgilio F. J. Immunol. 1997; 159: 1451-1458PubMed Google Scholar, Gabel C.A. J. Immunol. 1998; Google Scholar). The of the release and processing by ATP a of the that the processing and release of IL-1β and In this context, we this of ATP to compare IL-18 and IL-1β processing and release. We that are IL-1β and IL-18 in this and we the constitutive of to the role of priming in the release of we show that, although IL-18 is present in fresh the of ATP alone is insufficient to IL-18 release. specific for the and precursor of these we that the ICE inhibitor, the ATP-induced of both IL-1β and IL-18 but not release. we show that exogenous ATP rapidly caspase-1 in both and monocytes. this ATP effect is present and functional P2X7 Finally, we show that the ATP-induced activation at in protein-tyrosine kinase since it is by the but not by inhibition by mitogen-activated protein kinase, PKA we that both IL-1β and IL-18 are released in a similar fashion and that LPS priming is for this activation for IL-18 and by for IL-1β as we the report that caspase-1 activation is regulated by a protein-tyrosine kinase The for LPS priming for the ATP of IL-18 was and is particularly LPS priming was for the of intracellular IL-1β, we that the ATP effect was a trigger of the ICE activation and protein release However, the with IL-18 the to suggest that ATP alone is not to trigger processing release. LPS was not to an of P2X7 since our and constitutive of P2X7 receptors with only by LPS The role of LPS priming in caspase-1 activation is of shown in ATP stimulation alone caspase-1 without processing of constitutive IL-18 its from the and However, ATP stimulation after LPS priming both caspase-1 activation and IL-18 processing and release and suggest that the ATP effect on the release pathway is from its effect on ICE activation. In of we it to the of IL-1β IL-18 monocytes A data suggest that the processing and release of IL-1β and IL-18 are and However, these are of since the caspase-1 inhibitor, not the release of the precursor these do not the that caspase-1 a role in release that is from its as a in ICE is a in release of both the and precursor of IL-1β P. Allen H. Banerjee S. S. Herzog L. C. J. Paskind M. L. J. E. Tracey D. S. Wong W. Kamen R. T. 1995; Full Text PDF PubMed Scopus Google Scholar). the that caspase-1 molecules of the protein involved in IL-1β and IL-18 release. caspase-1 a classic can that molecules of this release M. K. K. F. C. J. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). The induced by similar domains are to be important in the of P. J. J. G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). it has been that human monocytes do not express P2X7 we were by the of fresh monocytes to ATP in our (20Dubyak G.R. El-Moatassim C. Am. J. Physiol. 1993; 265: C577-C606Crossref PubMed Google Scholar). and that monocytes express P2X7 receptors and the of LPS only induced P2X7 receptor The role of the P2X7 was by of oxidized ATP, a of completely and blocked the ATP-mediated release of IL-1β in monocytes. In the effect was by a monoclonal antibody at the P2X7 receptor but not by an Finally, the of P2X7 receptors was shown by both and by monocytes are to ATP-induced IL-1β activation via P2X7 pathways We that activation of protein kinases a role in release of IL-1β and classic protein kinase we for kinases that We were by the inhibition by the is a protein-tyrosine kinase that has been to be of in a A. A. M. A. N. A. Science. 1994; PubMed Scopus Google Scholar). is particularly from the that the effect that in ICE both ICE processing and IL-1β and IL-18 release. inhibition the in from P. Allen H. Banerjee S. S. Herzog L. C. J. Paskind M. L. J. E. Tracey D. S. Wong W. Kamen R. T. 1995; Full Text PDF PubMed Scopus Google Scholar). Although IL-18 and IL-1β are not from ICE are P. Allen H. Banerjee S. S. Herzog L. C. J. Paskind M. L. J. E. Tracey D. S. Wong W. Kamen R. T. 1995; Full Text PDF PubMed Scopus Google Scholar). at the caspase-1 be In this context, it is important to that inhibition of ICE by does not release, whereas of caspase-1 with can that are molecules that are to caspase-1. it that an aspect of caspase-1 of its is to release. both caspase-1 the of this kinase a activation step in IL-1β/IL-18 processing and release, to be to the response In an to the present in context, it be to the of our results to the signaling pathways for ATP and ATP in in and activation in cells N. J.M. M. 1998; PubMed Scopus Google Scholar, D. S. K. J. Biol. 1997; PubMed Scopus Google Scholar, Dubyak G.R. J. Immunol. 1996; Google Scholar). LPS is to activation of and protein kinase signaling pathways M.J. J. Biol. 1996; PubMed Scopus Google Scholar). LPS activation of family of kinases and However, these of family of kinases do not to be the of the and of IL-1β, and in lacking and is F. C.A. J. 1997; PubMed Scopus Google Scholar). In is not required for the release of IL-1β and IL-18, since and not processing and release but block the protein-tyrosine kinase by Taken these suggest that the regulation of the processing and release of IL-1β and IL-18 is similar and to the in these suggest that that this is to caspase-1 activation. the that the processing and release of IL-1β and IL-18 are to be similar and to be to regulation of We Dr. Ian for monoclonal antibody to P2X7 and Dr. Miller and Dr. Thornberry of for recombinant caspase-1 and caspase-1 We for the of
Mehta et al. (Thu,) studied this question.