Involvement of Caspase-1 and Caspase-3 in the Production and Processing of Mature Human Interleukin 18 in Monocytic THP.1 Cells

Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence was generated. However, natural hIL-18 has not yet been isolated, and its cellular processing is therefore still unclear. To clarify this, we purified natural hIL-18 from the cytosolic extract of monocytic THP.1 cells. Natural hIL-18 exhibited a molecular mass of 18.2 kDa, and the NH2-terminal amino acid was Tyr37. Biological activities of the purified protein were identical to those of recombinant hIL-18 with respect to the enhancement of natural killer cell cytotoxicity and interferon-γ production by human peripheral blood mononuclear cells. We also found two precursor hIL-18 (prohIL-18)-processing activities in the cytosol of THP.1 cells. These activities were blocked separately by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. Further analyses of the partially purified enzymes revealed that one is caspase-1, which cleaves prohIL-18 at the Asp36-Tyr37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp71-Ser72 and Asp76-Asn77 to generate biologically inactive products. These results suggest that the production and processing of natural hIL-18 are regulated by two processing enzymes, caspase-1 and caspase-3, in THP.1 cells. Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence was generated. However, natural hIL-18 has not yet been isolated, and its cellular processing is therefore still unclear. To clarify this, we purified natural hIL-18 from the cytosolic extract of monocytic THP.1 cells. Natural hIL-18 exhibited a molecular mass of 18.2 kDa, and the NH2-terminal amino acid was Tyr37. Biological activities of the purified protein were identical to those of recombinant hIL-18 with respect to the enhancement of natural killer cell cytotoxicity and interferon-γ production by human peripheral blood mononuclear cells. We also found two precursor hIL-18 (prohIL-18)-processing activities in the cytosol of THP.1 cells. These activities were blocked separately by the caspase inhibitors Ac-YVAD-CHO and Ac-DEVD-CHO. Further analyses of the partially purified enzymes revealed that one is caspase-1, which cleaves prohIL-18 at the Asp36-Tyr37 site to generate the mature hIL-18, and the other is caspase-3, which cleaves both precursor and mature hIL-18 at Asp71-Ser72 and Asp76-Asn77 to generate biologically inactive products. These results suggest that the production and processing of natural hIL-18 are regulated by two processing enzymes, caspase-1 and caspase-3, in THP.1 cells. Interleukin (IL) 1The abbreviations used are: IL, interleukin; pro, precursor; h, human; ICE, interleukin-1β-converting enzyme; mAb, monoclonal antibody; pAb, polyclonal antibody; PCR, polymerase chain reaction; PB, phosphate buffer; IFN, interferon; NK cell, natural killer cell; ICA, human IL-18-converting activity; IDA, human IL-18-degrading activity; hIL-18-CE, human IL-18-converting enzyme; hIL-18-DE, human IL-18-degrading enzyme; CHAPS, 3-(3-cholamidpropyl)dimethylammonio-1-propanesulfonic acid; DTT, dithiothreitol; Bis-Tris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-propane-1,3-diol; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PBMC, peripheral blood mononuclear cells. -18 (originally called IGIF, interferon-γ-inducing factor) is a novel cytokine with multiple biological functions. In 1995 we purified murine IL-18 from the liver extracts of mice sensitized withPropionibacterium acnes followed by elicitation with lipopolysaccaride (1Okamura H. Nagata K. Komatsu T. Tanimoto T. Nukada Y. Tanabe F. Akita K. Torigoe K. Okura T. Fukuda S. Kurimoto M. Infect. Immun. 1995; 63: 3966-3972Crossref PubMed Google Scholar). The cDNA of murine IL-18 was cloned from cDNA libraries prepared from the livers of mice with endotoxin shock (2Okamura H. Tsutsui H. Komatsu T. Yutsudo M. Hakura A. Tanimoto T. Torigoe K. Okura T. Nukada Y. Hattori K. Akita K. Namba M. Tanabe F. Konishi K. Fukuda S. Kurimoto M. Nature. 1995; 378: 88-91Crossref PubMed Scopus (2432) Google Scholar). Using this as a probe, human IL-18 cDNA was also cloned from a human normal liver cDNA library (3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar). The recombinant human IL-18 with a tentatively assigned NH2-terminal amino acid based on its homology with the natural murine IL-18 sequence was expressed in Escherichia coli, and its biological activities were examined (3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar). IL-18 has an interleukin 1 (IL-1) signature-like sequence (3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar) as reported and is similar to the IL-1 family and fibroblast growth factor in terms of their trefoil structures (4Bazan J.F. Timans J.C. Kastelein R.A. Nature. 1996; 379: 591Crossref PubMed Scopus (265) Google Scholar, 5Murzin A.G. Lesk A.M. Chothia C. J. Mol. Biol. 1992; 223: 531-543Crossref PubMed Scopus (307) Google Scholar). Despite their similarities, IL-18 and IL-1β exhibit different biological activities (2Okamura H. Tsutsui H. Komatsu T. Yutsudo M. Hakura A. Tanimoto T. Torigoe K. Okura T. Nukada Y. Hattori K. Akita K. Namba M. Tanabe F. Konishi K. Fukuda S. Kurimoto M. Nature. 1995; 378: 88-91Crossref PubMed Scopus (2432) Google Scholar, 3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar, 6Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar), transmitted through their specific receptors. 2Torigoe, K., Ushio, S., Okura, T., Kobayashi, S., Taniai, M., Kunikata, T., Murakami, T., Sanou, O., Kojima, H., Fujii, M., Ohta, T., Ikeda, M., Ikegami, H., and Kurimoto, M. (1997) J. Biol. Chem., in press. Genetic information suggested that IL-18 is synthesized as an inactive precursor form (prohIL-18) and that this prohIL-18 has no known signal peptide sequence. Therefore, proteolytic cleavage is required for its maturation like IL-1β (2Okamura H. Tsutsui H. Komatsu T. Yutsudo M. Hakura A. Tanimoto T. Torigoe K. Okura T. Nukada Y. Hattori K. Akita K. Namba M. Tanabe F. Konishi K. Fukuda S. Kurimoto M. Nature. 1995; 378: 88-91Crossref PubMed Scopus (2432) Google Scholar, 3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar, 7Gu Y. Kuida K. Tsutsui H. Ku G. Hsiao K. Fleming M.A. Hayashi N. Higashino K. Okamura H. Nakanishi K. Kurimoto M. Tanimoto T. Flavell R.A. Sato V. Harding M.W. Livingston D.J. Su M.S.-S. Science. 1997; 275: 206-209Crossref PubMed Scopus (1031) Google Scholar, 8Ghayur T. Banerjee S. Hugunin M. Butler D. Herzog L. Carter A. Quintal L. Sekut L. Talanian R. Paskind M. Wong W. Kamen R. Tracey D. Allen H. Nature. 1997; 386: 619-623Crossref PubMed Scopus (1055) Google Scholar). Gu et al. (7Gu Y. Kuida K. Tsutsui H. Ku G. Hsiao K. Fleming M.A. Hayashi N. Higashino K. Okamura H. Nakanishi K. Kurimoto M. Tanimoto T. Flavell R.A. Sato V. Harding M.W. Livingston D.J. Su M.S.-S. Science. 1997; 275: 206-209Crossref PubMed Scopus (1031) Google Scholar) reported that IL-1β-converting enzyme (ICE)/caspase-1 cleaved murine proIL-18 at the authentic processing site, Asp35-Asn36, to generate biologically active mature murine IL-18. However, natural hIL-18 had not yet been isolated, and its maturation site remained unclear. In this report, we screened for hIL-18 mRNA-expressing cell lines and purified natural hIL-18 from the cell-free extract of positively expressing cells. Furthermore, we identified two hIL-18-processing enzymes in the same cellular extract. One enzyme is ICE/caspase-1, which acts on prohIL-18 to generate the mature active form of hIL-18, and the other is CPP32/caspase-3, which acts on both the prohIL-18 and the mature hIL-18 to generate biologically inactive degraded products. This is the first report on the identification of natural hIL-18 and its processing enzymes existing in the same cells. Tetrapeptidyl ICE-like protease inhibitor Ac-YVAD-CHO and CPP32-like protease inhibitor Ac-DEVD-CHO, and ICE- and CPP32-like protease fluorogenic substrates Ac-YVAD-MCA and Ac-DEVD-MCA, respectively, were purchased from the Peptide Institute (Osaka, Japan). Neutralizing and non-neutralizing anti-hIL-18 murine monoclonal antibodies (mAbs) 125-2H (IgG) and 25-2G (IgG) were raised against the recombinant protein in our laboratory. Human IL-1β precursor and anti-precursor human IL-1β rabbit polyclonal antibody (pAb) were obtained from Cistron (Pine Brook, NJ). Anti-human ICE-p20 subunit, anti-human CPP32, anti-human poly(ADP-ribose)polymerase goat pAbs, and anti-human ICE-p10 subunit rabbit pAb were purchased from Santa Cruz purified human CPP32, and rabbit were prepared at cell lines were for the of hIL-18 in with and were in a and at To of THP.1 cell was as S. T. Tanimoto T. H. Torigoe K. Kurimoto M. J. PubMed Scopus Google Scholar, S. S. M. Fujii M. N. K. Torigoe K. T. Kurimoto M. Scholar). THP.1 were and with of a rabbit the were of the the of THP.1 were with and were by and through to a cell were from cell lines by in with the The were and by The were of for 1 for 1 and for 1 for and The of the for hIL-18 were and which a The were on and with were at for which was at THP.1 cell-free extract was prepared by G. J. A.G. S. A. PubMed Scopus Google Scholar). the cell from THP.1 were with of and were by at for The cell were in of and at were by of and cell was by at The cell-free extract obtained was against phosphate at The cell-free extract from 1 was a with at with the same the extract was with and The hIL-18 with were and to with a and against at The from was through a a with at and with the same was with of a and were hIL-18 was at in PB, and the were and to with hIL-18 was prepared and anti-hIL-18 murine in our laboratory. The and from was a hIL-18 with at with the same the was with at were with The from were a with and the was with the same the was with of an The at and was used for peptide of natural production and NK cell cytotoxicity were as (3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar). in the were at for which was at THP.1 were obtained as extract was prepared by the of et al. A.M. J. G. R.A. J. Biol. PubMed Google Scholar). was to the extract to The was for and the was by The was to with and the by was in CHAPS, and against the same for The from was through a of the was a with and with the same The extract was in two of with of a at and at were separately and against CHAPS, These two were to of the was a with and with the same the was in two of with of a at in and were and against This was with a in with the of the was a with and with the same the was in of with an were and used in the of the from was a with The was in of with the same was in active were and to NH2-terminal of the of the was a with at and with the same The was in of with were and against at of the from 1 was a with at and with the same The was in two of with and acid at which a were and against The from were and a with at was in were and used in The from were in two a with at The was in with of a phosphate at phosphate in in and which were and to NH2-terminal of the acid of the NH2-terminal of purified natural hIL-18 and of hIL-18 and and of partially purified hIL-18-processing enzymes were with an protein of were to in and The were by and for NH2-terminal peptide purified natural hIL-18 was with and the peptide were a with of in at a of were separately and cDNA precursor hIL-18 (3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar) was the H. A. F. J. PubMed Scopus Google Scholar). The hIL-18 was by and were The expressed protein was by a Human cDNA was cloned from THP.1 the were based on the sequence J. J. G. A.M. M. R.A. Nature. 1992; PubMed Scopus Google Scholar). were by with an cDNA and from the were with a The of this was at for to precursor and was partially purified by followed by and The of hIL-18 were by a prepared in our M. K. T. T. H. S. Ikeda M. Kurimoto M. J. Immunol. 1997; Scholar). The of hIL-18 was by its on the cell F. M. H. T. M. Ikeda, K. and M. Kurimoto, for in were in a were to the and for h, and in the was by ICE-like and CPP32-like activities were the fluorogenic substrates Ac-YVAD-MCA and Ac-DEVD-MCA, A.M. J. G. R.A. J. Biol. PubMed Google J. J. G. A.M. M. R.A. 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Nature. 1995; PubMed Scopus Google Scholar). 1 and were at in the of and in at for at for The of were with a at a of for and for The activities of were and as of To cell we first examined the of of hIL-18 by cell lines of were hIL-18 was expressed in cell lines as and and and cell lines as and In cell and cell lines as and of hIL-18 was no was in cell lines of hIL-18 in cell cell cell cell cell cell of of hIL-18 were examined by cell lines of were The of was from the of the no in a The of of hIL-18 were examined by cell lines of were The of was from the of the no we cell lines and and two cell lines and as for the of natural hIL-18 at the protein a extracts of cell lines were and of hIL-18 were in the cell extracts of THP.1 and no hIL-18 was in the not Furthermore, hIL-18 was found in the cytosolic of the cells. we and to the production of However, no in the of hIL-18 was not the we the cytosolic extract of THP.1 as a for the of natural this of THP.1 were obtained with an in cell S. T. Tanimoto T. H. Torigoe K. Kurimoto M. J. PubMed Scopus Google Scholar, S. S. M. Fujii M. N. K. Torigoe K. T. Kurimoto M. Scholar), and a cytosolic extract was prepared by The cell extract was to and for the of natural on anti-hIL-18 was The purified protein had a molecular mass of 18.2 on and exhibited on 1 The of natural hIL-18 from to cells. amino acid of the that the NH2-terminal amino acid is Tyr37. Furthermore, the amino acid of the prepared from the purified with those from the cDNA These results that the purified from THP.1 is a mature form of hIL-18 the in a similar to that of the murine (1Okamura H. Nagata K. Komatsu T. Tanimoto T. Nukada Y. Tanabe F. Akita K. Torigoe K. Okura T. Fukuda S. Kurimoto M. Infect. Immun. 1995; 63: 3966-3972Crossref PubMed Google Scholar, H. Tsutsui H. Komatsu T. Yutsudo M. Hakura A. Tanimoto T. Torigoe K. Okura T. Nukada Y. Hattori K. Akita K. Namba M. Tanabe F. Konishi K. Fukuda S. Kurimoto M. 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The enhancement of NK cell cytotoxicity was at as as These results suggest that the purified natural hIL-18 is to the recombinant protein in its biological functions. the natural hIL-18, the THP.1 cell-free extract also the precursor protein as by not This that proteolytic maturation in THP.1 in the cell-free extract the The amino acid sequence of the hIL-18 maturation site to specific to Livingston D.J. G. Wong J. 1996; 87: PubMed Scopus Google Scholar) Nature. 1995; PubMed Scopus Google Scholar), the of an IL-18-converting enzyme in the cytosolic extract of THP.1 cells. To this, the recombinant precursor protein of hIL-18 was for with the THP.1 cell extract. antibody revealed that prohIL-18 was cleaved by the THP.1 extracts to generate cleavage with molecular of 18 and the first and and in a of the prohIL-18 and in a of the In an in the of the and was of and was that are at cleavage in prohIL-18 as substrates for the processing In the same hIL-18 activities were by a M. K. T. T. H. S. Ikeda M. Kurimoto M. J. Immunol. 1997; Scholar) and by on in to hIL-18, not to hIL-18 with the of the and were by the and no on to proteolytic cleavage is by family by protease inhibitors were of The of the was by a Ac-YVAD-CHO the of and had no on that of In to this, the of and had no on that of These results suggest that are at two enzymes in THP.1 cell one is an ICE-like protease the mature form of hIL-18, and the other is a CPP32-like protease inactive degraded of To the we to the two hIL-18-processing and from the cytosolic extract of THP.1 cells. were partially based on those used for the of A.M. J. G. R.A. J. Biol. PubMed Google Scholar, J. J. G. A.M. M. R.A. Nature. 1992; PubMed Scopus Google Scholar). activities were by at a at a as a We also examined the ICE-like and CPP32-like activities J. J. G. A.M. M. R.A. Nature. 1992; PubMed Scopus Google Scholar, A. M. Y. M. Nature. 1995; PubMed Scopus Google Scholar) in the the fluorogenic substrates and The of activities of two fluorogenic with those of and as by in prohIL-18 cleavage of also exhibited cleavage This cleavage of by was purified of not Therefore, for of and IDA, we both ICE-like and CPP32-like cleavage on fluorogenic substrates and the in cleavage was partially purified by followed by not The from were to NH2-terminal amino acid sequence this both the and of and were at an The of two in the was also by and not This suggested that enzyme To this in cleavage of precursor human IL-1β by partially purified were precursor human IL-1β to generate the mature IL-1β as as the mature hIL-18 These cleavage activities were by Ac-YVAD-CHO and The of the mature was by for hIL-18 and S. A. PubMed Scopus Google Scholar) for IL-1β not Furthermore, recombinant cleaved prohIL-18 to generate the mature hIL-18 These results the that enzyme is amino acid of hIL-18-processing enzymes in partially purified acid amino and were in the first of the partially purified and were to followed by NH2-terminal as The a that not assigned based on peptide sequence amino and were in the first of the in a The partially purified and were to followed by NH2-terminal as The a that not assigned based on peptide sequence enzyme was partially purified by followed by not of the those of the are reported to poly(ADP-ribose)polymerase A. M. Y. M. Nature. 1995; PubMed Scopus Google Scholar, T. G. 1995; Google Scholar, Gu Y. C. Su M.S.-S. J. Biol. 1996; PubMed Scopus Google Scholar). Therefore, we this cleavage partially purified THP.1 cleaved poly(ADP-ribose)polymerase to generate an NH2-terminal as as degraded prohIL-18 The partially purified was also to NH2-terminal amino acid sequence this both the and of and and the subunit of were The of two enzymes were to analyses also that both and in the not These results suggested that the for the enzyme was recombinant enzymes that of prohIL-18 is by not by cleaved prohIL-18 and at and not we that found in THP.1 cell is Natural hIL-18 and its processing enzymes were identified in the cytosolic extract of monocytic THP.1 cells. has been reported that murine IL-18 is by (2Okamura H. Tsutsui H. Komatsu T. Yutsudo M. Hakura A. Tanimoto T. Torigoe K. Okura T. Nukada Y. Hattori K. Akita K. Namba M. Tanabe F. Konishi K. Fukuda S. Kurimoto M. Nature. 1995; 378: 88-91Crossref PubMed Scopus (2432) Google Scholar) M. Y. T. Ikeda M. Kurimoto M. 1997; PubMed Scopus Google Scholar). are to to murine IL-18 with the of (7Gu Y. Kuida K. Tsutsui H. Ku G. Hsiao K. Fleming M.A. Hayashi N. Higashino K. Okamura H. Nakanishi K. Kurimoto M. Tanimoto T. Flavell R.A. Sato V. Harding M.W. Livingston D.J. Su M.S.-S. Science. 1997; 275: 206-209Crossref PubMed Scopus (1031) Google Scholar). the IL-18 is expressed in the of IL-18 to H., K., H., K., and K. in In our to cell of hIL-18 in cell lines was of were in and cell cell and lines of IL-18 cell lines not at These results the that are cells. of natural hIL-18, precursor protein degraded were by from which the mature hIL-18 was by specific 1 In respect of the enhancement of NK cell cytotoxicity and production by human PBMC, purified natural hIL-18 the same biological as those of the recombinant protein that had an NH2-terminal sequence tentatively assigned based on homology with the murine natural IL-18 In the maturation site of natural hIL-18 was in with that of the murine hIL-18 has no site, and the molecular mass from the information is The molecular mass of natural hIL-18 by is 18.2 1 These results suggest that natural hIL-18 is a protein a chain Natural prohIL-18 is as a by the molecular mass of prohIL-18 is that the of natural prohIL-18 is blocked by from the that the NH2-terminal amino acid not by the not These results suggest that prohIL-18 to its in the This that specific active in to mature IL-18 the to However, on hIL-18 mRNA-expressing cell lines to hIL-18 the same that IL-1β is not This to the of of the precursor protein in the is that the for different hIL-18 and The is by the on the of IL-1β by and J. Biol. 1995; PubMed Scopus Google Scholar). respect to the IL-18 and the IL-1 signature-like sequence (3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar) and the of been reported (4Bazan J.F. Timans J.C. Kastelein R.A. Nature. 1996; 379: 591Crossref PubMed Scopus (265) Google Scholar). of in the maturation of hIL-18 has also been in this However, the biological of IL-18 from those of IL-1β in terms of the of NK cell cytotoxicity and and factor production by and the of cell (3Ushio S. Namba M. Okura T. Hattori K. Nukada Y. Akita K. Tanabe F. Konishi K. Micallef M. Fujii M. Torigoe K. Tanimoto T. Fukuda S. Ikeda M. Okamura H. Kurimoto M. J. Immunol. 1996; 156: 4274-4279PubMed Google Scholar, T. K. Tanabe F. S. Namba M. Tanimoto T. Torigoe K. Fujii M. Ikeda M. Fukuda S. Kurimoto M. J. 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G. 1995; Google Scholar) in cleavage mature hIL-18 revealed that cleaved not prohIL-18 also mature hIL-18 as a to generate the same biologically inactive and not These results suggest that as a of IL-18. We in in Further are therefore to processing for IL-18. However, is that proIL-18 and with other as substrates for active is reported that to as an inhibitor against cleavage of other by in T. J. J. Immunol. 1996; Scholar). proIL-18 with in an In of ICE-like and CPP32-like enzymes in murine M. Talanian Wong Nagata S. Nature. 1996; PubMed Scopus Google Scholar). that prohIL-18 mature hIL-18 also to the of The that two and which the for IL-18 is a for on of IL-18. In the analyses on natural hIL-18 purified from monocytic THP.1 revealed the of in its was found to hIL-18 to biologically inactive are to the of to on the of IL-18 in We Micallef for a of the and this We also for anti-hIL-18 and and Taniai, Ushio, and for