Interleukin-33 Is Biologically Active Independently of Caspase-1 Cleavage

Abstract

The new interleukin (IL)-1 family cytokine IL-33 is synthesized as a 30-kDa precursor. Like pro-IL-1β, human pro-IL-33 was reported to be cleaved by caspase-1 to generate an 18-kDa fragment, which is sufficient to activate signaling by the IL-33 receptor T1/ST2. However, the proposed caspase-1 cleavage site is poorly conserved between species. In addition, it is not clear whether caspase-1 cleavage of pro-IL-33 occurs in vivo and whether, as for IL-1β, this cleavage is a prerequisite for IL-33 secretion and bioactivity. In this study, we further investigated caspase-1 cleavage of mouse and human pro-IL-33 and assessed the potential bioactivity of the IL-33 precursor. We observed the generation of a 20-kDa IL-33 fragment in cell lysates, which was enhanced by incubation with caspase-1. However, in vitro assays of mouse and human pro-IL-33 indicated that IL-33 is not a direct substrate for this enzyme. Consistently, caspase-1 activation in THP-1 cells induced cleavage of pro-IL-1β but not of pro-IL-33, and activated THP-1 cells released full-length pro-IL-33 into culture supernatants. Finally, addition of full-length pro-IL-33 induced T1/ST2-dependent IL-6 secretion in mast cells. However, we observed in situ processing of pro-IL-33 in mast cell cultures, and it remains to be determined whether full-length pro-IL-33 itself indeed represents the bioactive species. In conclusion, our data indicate that pro-IL-33 is not a direct substrate for caspase-1. In addition, our results clearly show that caspase-1 cleavage is not required for pro-IL-33 secretion and bioactivity, highlighting major differences between IL-1β and IL-33. The new interleukin (IL)-1 family cytokine IL-33 is synthesized as a 30-kDa precursor. Like pro-IL-1β, human pro-IL-33 was reported to be cleaved by caspase-1 to generate an 18-kDa fragment, which is sufficient to activate signaling by the IL-33 receptor T1/ST2. However, the proposed caspase-1 cleavage site is poorly conserved between species. In addition, it is not clear whether caspase-1 cleavage of pro-IL-33 occurs in vivo and whether, as for IL-1β, this cleavage is a prerequisite for IL-33 secretion and bioactivity. In this study, we further investigated caspase-1 cleavage of mouse and human pro-IL-33 and assessed the potential bioactivity of the IL-33 precursor. We observed the generation of a 20-kDa IL-33 fragment in cell lysates, which was enhanced by incubation with caspase-1. However, in vitro assays of mouse and human pro-IL-33 indicated that IL-33 is not a direct substrate for this enzyme. Consistently, caspase-1 activation in THP-1 cells induced cleavage of pro-IL-1β but not of pro-IL-33, and activated THP-1 cells released full-length pro-IL-33 into culture supernatants. Finally, addition of full-length pro-IL-33 induced T1/ST2-dependent IL-6 secretion in mast cells. However, we observed in situ processing of pro-IL-33 in mast cell cultures, and it remains to be determined whether full-length pro-IL-33 itself indeed represents the bioactive species. In conclusion, our data indicate that pro-IL-33 is not a direct substrate for caspase-1. In addition, our results clearly show that caspase-1 cleavage is not required for pro-IL-33 secretion and bioactivity, highlighting major differences between IL-1β and IL-33. Interleukin (IL) 2The abbreviations used are: ILinterleukinBMMCbone marrow-derived mast cell(s)CHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidGFPgreen fluorescent proteinhhumanLPSlipopolysaccharidemmousePMAphorbol 12-myristate 13-acetatepro-ILprointerleukinrrecombinantssoluble. -33, the most recently described cytokine of the IL-1 family, is synthesized as a 30-kDa precursor. Human pro-IL-33, like pro-IL-1β, was reported to be cleaved by caspase-1 in vitro to generate an 18-kDa fragment, termed mature IL-33, which is sufficient to activate signaling by the IL-33 receptor T1/ST2 (1.Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2968) Google Scholar). interleukin bone marrow-derived mast cell(s) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid green fluorescent protein human lipopolysaccharide mouse phorbol 12-myristate 13-acetate prointerleukin recombinant soluble. Caspase-1 is an endoproteinase that specifically cleaves Asp-Xaa bonds, where Xaa typically refers to a small, often hydrophobic residue (2.Howard A.D. Kostura M.J. Thornberry N. Ding G.J. 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, 3.Lüschen S. Ussat S. Krönke M. Adam-Klages S. Biochem. Biophys. Res. Commun. 1998; 253: 92-98Crossref PubMed Scopus (31) Google Scholar, 4.Lamkanfi M. Kanneganti T.D. Van Damme P. Vanden Berghe T. Vanoverberghe I. Vandekerckhove J. Vandenabeele P. Gevaert K. Núñez G. Mol. Cell. Proteomics. 2008; 7: 2350-2363Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Caspase-1 activity absolutely requires the presence of an Asp residue at position −1 of the cleavage site. Consistently, replacement of Asp116 by other amino acids, such as Ala, was previously demonstrated to prevent caspase-1 cleavage of pro-IL-1β (2.Howard A.D. Kostura M.J. Thornberry N. Ding G.J. 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). Recombinant (r) mature IL-33 starts at Ser112 for human (h) IL-33 and at Ser109 for mouse (m) IL-33, neither of which corresponds exactly to the position of a potential caspase-1 cleavage site. Indeed, the N-terminal moiety of human pro-IL-33 sequence contains a single Asp at position 110, and the N-terminal portion of mouse pro-IL-33 contains an Asp at positions 88 and 106. In fact, the region located between amino acids 80 and 110 of pro-IL-33 is rather poorly conserved between species (5.Carriere V. Roussel L. Ortega N. Lacorre D.A. Americh L. Aguilar L. Bouche G. Girard J.P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 282-287Crossref PubMed Scopus (818) Google Scholar). In particular, no Asp residues can be consistently found at an identical position across species to hint at the presence of a conserved caspase-1 cleavage site. So far, caspase-1 cleavage of pro-IL-33 has not been investigated in any species other than human. Expression of endogenous IL-33 has been described most extensively in endothelial cells, where essentially nuclear, full-length 30-kDa pro-IL-33 is detected (5.Carriere V. Roussel L. Ortega N. Lacorre D.A. Americh L. Aguilar L. Bouche G. Girard J.P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 282-287Crossref PubMed Scopus (818) Google Scholar, 6.Küchler A.M. Pollheimer J. Balogh J. Sponheim J. Manley L. Sorensen D.R. De Angelis P.M. Scott H. Haraldsen G. Am. J. Pathol. 2008; 173: 1229-1242Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 7.Moussion C. Ortega N. Girard J.P. PLoS ONE. 2008; 3: e3331Crossref PubMed Scopus (939) Google Scholar). To date, only two studies have examined potential effects of caspase-1 activation on the processing and secretion of pro-IL-33 in living cells. In one study, stimulation of murine glial cultures with caspase-1 activators induced secretion of bioactive IL-33 into culture supernatants, but the size of the secreted protein was not assessed (8.Hudson C.A. Christophi G.P. Gruber R.C. Wilmore J.R. Lawrence D.A. Massa P.T. J. Leukocyte Biol. 2008; 84: 631-643Crossref PubMed Scopus (149) Google Scholar). It is thus not clear whether caspase-1 cleavage of pro-IL-33 occurs in mouse cells. In a second study, Western blot analysis revealed the presence of a 32-kDa protein and minor 17 and 20 kDa bands reacting with anti-IL-33 antibodies in the supernatants of THP-1 cells upon caspase-1 activation, suggesting secretion of full-length pro-IL-33 and of two potential cleavage products (9.Li H. Willingham S.B. Ting J.P. Re F. J. Immunol. 2008; 181: 17-21Crossref PubMed Scopus (33) Google Scholar). Although this last observation suggests that some pro-IL-33 may be secreted, it not known to what extent IL-33 secretion is dependent on caspase-1 cleavage. Finally, so far all studies reporting T1/ST2-mediated effects of IL-33 were performed using the recombinant mature form of IL-33, whereas potential biological activity of the full-length precursor form has not been tested. It thus remains to be shown whether, as for IL-1β, caspase-1 cleavage is indeed required for IL-33 bioactivity. In the present study, we thus further investigated caspase-1 cleavage of mouse and human pro-IL-33 in vitro and in cultured cells and assessed the potential bioactivity of the IL-33 precursor. Cell culture reagents were obtained from Invitrogen. rmIL-33 and rhIL-33 produced in Escherichia coli were obtained from Alexis Corp. (Lausen, Switzerland). Soluble (s) ST2-Fc fusion protein was obtained from Amgen (Thousand Oaks, CA). T1/ST2 knock-out BALB/c mice were generously provided by Dr. A. McKenzie (Medical Research Council, Cambridge, UK). An IMAGE expressed sequence tag clone containing the cDNA encoding the full-length mouse pro-IL-33 (GenBankTM accession number BC003847) in the pCMV-Sport6 vector was used as expression vector for full-length mouse pro-IL-33 in 293T human embryonic kidney cells. Mutagenesis of Asp to Ala in mouse D88A and D106A pro-IL-33 mutants was performed using the QuikChange kit (Stratagene, La Jolla, CA) and appropriate primer pairs, according to the manufacturer's instructions. The cDNA corresponding to the full-length human pro-IL-33 (GenBankTM accession number AY905581) was amplified by reverse transcription-PCR using total RNA extracted from cultured rheumatoid arthritis synovial fibroblasts (forward primer, 5′-cgggatccatgaagcctaaaatgaagtattc-3′; and reverse primer, 5′-gctctagactaagtttcagagagcttaaac-3′) and cloned into the pCAGGSβ vector (10.Gabay C. Gigley J. Sipe J. Arend W.P. Fantuzzi G. Eur. J. Immunol. 2001; 31: 490-499Crossref PubMed Scopus (51) Google Scholar) for expression in 293T cells. For expression in THP-1 cells, full-length human pro-IL-33 cDNA was cloned into the pWPI lentiviral vector (11.Wiznerowicz M. Trono D. J. Virol. 2003; 77: 8957-8961Crossref PubMed Scopus (635) Google Scholar). Viral particles were produced in 293T cells using a standard protocol as described (12.Dunand-Sauthier I. Hohl M. Thorel F. Jaquier-Gubler P. Clarkson S.G. Schärer O.D. J. Biol. Chem. 2005; 280: 7030-7037Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The cDNA corresponding to full-length human pro-IL-1β (GenBankTM accession number NM_000576) was amplified by reverse transcription-PCR using RNA extracted from phorbol 12-myristate 13-acetate (PMA)-stimulated THP-1 cells (forward primer, 5′-cgggatccggctgctctgggattctcttcag-3′; and reverse primer, 5′-gcctcgaggggtacagctctctttaggaag-3′) and cloned into the pcDNA3.1+ vector (Invitrogen) for expression in 293T cells. The sequence of the entire coding region was verified before use for all constructs. Human embryonic kidney 293T cells were cultured in Dulbecco's modified Eagle's medium (4.5 g/liter glucose) supplemented with l-glutamine, streptomycin, penicillin, and 10% fetal calf serum at 37 °C in a humidified atmosphere containing 5% CO2. For transfection, 293T cells were plated at a density of 100,000 cells/ml and transfected 24 h after seeding with 1 μg/ml pro-IL-33 or pro-IL-1β expression vector, or empty vectors as a control, by calcium phosphate precipitation (13.Palmer G. Trolliet S. Talabot-Ayer D. Mézin F. Magne D. Gabay C. Cytokine. 2005; 31: 153-160Crossref PubMed Scopus (25) Google Scholar). THP-1 cells were cultured in RPMI 1640 medium (4.5 g/liter glucose) supplemented with 10 mm Hepes, 1 mm sodium pyruvate, 5 × 10−5m β-mercaptoethanol, streptomycin, penicillin, and 10% fetal calf serum. For viral infection, THP-1 cells were plated at a density of 250,000 cells/well and infected with freshly produced lentiviruses encoding hIL-33 and green fluorescent protein (GFP), or GFP alone as a control, using a double infection protocol (12.Dunand-Sauthier I. Hohl M. Thorel F. Jaquier-Gubler P. Clarkson S.G. Schärer O.D. J. Biol. Chem. 2005; 280: 7030-7037Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). P815 cells were cultured in Dulbecco's modified Eagle's medium (4.5 g/liter glucose) supplemented with 6% fetal calf serum, 10 mm Hepes, 10 mm minimum Eagle's medium nonessential amino acids, penicillin, and streptomycin. Primary mouse bone marrow-derived mast cells (BMMC) were generated as described (14.Moulin D. Donzé O. Talabot-Ayer D. Mézin F. Palmer G. Gabay C. Cytokine. 2007; 40: 216-225Crossref PubMed Scopus (270) Google Scholar) from wild-type and T1/ST2 knock-out BALB/c mice (15.Townsend M.J. Fallon P.G. McKenzie J. PubMed Scopus Google Scholar) and used after 10 of and D88A and D106A mouse pro-IL-33 as as human pro-IL-33 and pro-IL-1β were synthesized in vitro using the in vitro protein kit kit contains a from and is thus essentially of any To generate for the in vitro protein the were amplified from the described expression vectors using appropriate to a and an E. coli site of the according to the manufacturer's instructions. The sequence of all products was verified before was using of in a of cDNA were as 293T cells were used h after were in mm Hepes, mm 10% 10 mm by on and at Cell were for h at or with of recombinant human caspase-1 Alexis In vitro synthesized were in 20 of for h at 37 °C or with of recombinant human caspase-1. were by the addition of and were on and by Western THP-1 cells were for h with in RPMI 1640 medium and to were to (Invitrogen) and or not with μg/ml lipopolysaccharide for as described previously V. S. C. A. F. J. Cell 2007; PubMed Scopus Google Scholar). supernatants were and cell were by after addition of and cell were by in by and by Western were by and to a The was in containing 5% serum or 5% and was performed with one of the as anti-IL-33 Alexis and or the for mature Cell bands were by using appropriate THP-1 cells were in with and or not with μg/ml as described The cells were 10 at 10 and serum 10% 1 before with a Alexis and with a To were performed in the of the were using a medium and were on an Switzerland). 293T cells were in and transfected with the mouse pro-IL-33 expression were h after in mm mm supplemented with a and by on transfected cells were used to standard J.F. L. J. Virol. PubMed Scopus Google Scholar). cells were in on and were in and by the addition of pro-IL-33 was from total cell or from using mouse and the protein kit and in using the acid provided in the were using of and used for Western after the addition of serum at were used to P815 cells or of mouse pro-IL-33 or of in vitro synthesized and D106A mouse pro-IL-33 was assessed using P815 cells and (14.Moulin D. Donzé O. Talabot-Ayer D. Mézin F. Palmer G. Gabay C. Cytokine. 2007; 40: 216-225Crossref PubMed Scopus (270) Google Scholar). P815 cells were plated in in of culture medium at a density of cells/well and with the indicated of rmIL-33 or with the indicated of in vitro products for 24 h before culture supernatants were To of the observed or in vitro synthesized were with for at 37 °C before addition to the cells. generated from wild-type or T1/ST2 knock-out mice were or with pro-IL-33 of of total rmIL-33 or for 24 h before culture supernatants were of IL-6 in P815 and culture supernatants were assessed using an kit The of differences was assessed by analysis of between was the was Human pro-IL-33, like pro-IL-1β, was reported to be cleaved by caspase-1 in vitro to generate an 18-kDa fragment (1.Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2968) Google Scholar). However, the proposed caspase-1 cleavage site is poorly conserved between species (5.Carriere V. Roussel L. Ortega N. Lacorre D.A. Americh L. Aguilar L. Bouche G. Girard J.P. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 282-287Crossref PubMed Scopus (818) Google Scholar). we further investigated caspase-1 cleavage of mouse and human pro-IL-33 was in 293T cells, and total cell were with recombinant human caspase-1 in Western blot analysis revealed the presence of a major corresponding to the 30-kDa full-length form of pro-IL-33, after incubation with caspase-1 In addition, we detected a at 20 the of which was in the presence of caspase-1. corresponding to the size of rmIL-33 was detected in the or presence of caspase-1. results were obtained upon incubation of the cell with recombinant mouse caspase-1 and using two antibodies and for and data not In an to which of the two N-terminal Asp residues a potential caspase-1 cleavage site in mouse pro-IL-33, we generated two of mouse pro-IL-33, D88A and in which or was by neither of two with the generation of the 20 kDa which was in cell and enhanced in the presence of caspase-1 of 293T cells human pro-IL-33 essentially the 30-kDa full-length form of pro-IL-33, after incubation with as as a minor at 20 kDa results were obtained using two antibodies and for and data not For and the observed 20-kDa fragment was of than 18-kDa 20 kDa was before caspase-1 was in cell 1 and data not However, the 30-kDa full-length pro-IL-33 the most form detected in all in the presence of caspase-1. Finally, of 293T cells pro-IL-1β were used as a for caspase-1 activity in our and pro-IL-1β was cleaved into mature form The presence of the 20 kDa in cell as as in cells transfected with pro-IL-33 that generation of this IL-33 fragment is not by caspase-1. In fact, 293T cell a number of that be in IL-33 We thus to caspase-1 cleavage of pro-IL-33 in a To this we synthesized pro-IL-33 using an in vitro from and recombinant which is essentially in vitro synthesized mouse or human pro-IL-33 with recombinant human we not any cleavage of pro-IL-33, which was in 30-kDa full-length after h of In using the human pro-IL-1β was cleaved to generate mature IL-1β in to pro-IL-1β, pro-IL-33 is not a direct substrate for caspase-1. We investigated the of caspase-1 activation on pro-IL-33 processing and secretion in living cells. For we used THP-1 cells caspase-1 activation and IL-1β secretion have been described extensively in cells, in to and in V. S. C. A. F. J. Cell 2007; PubMed Scopus Google Scholar). In our THP-1 cells not endogenous pro-IL-33 as assessed by reverse transcription-PCR and Western and data not We thus used a lentiviral vector to human pro-IL-33 in cells. lentiviral expression of pro-IL-33 was observed essentially in the of THP-1 cells, with and stimulation and data not We potential pro-IL-33 cleavage and secretion upon activation of endogenous caspase-1 in to in THP-1 cells V. S. C. A. F. J. Cell 2007; PubMed Scopus Google Scholar). and secretion of endogenous IL-1β were in in the as a Western blot analysis revealed the presence of 30-kDa full-length pro-IL-33 as as the 20-kDa fragment in cell of THP-1 cells with the IL-33 expression vector The of the 20-kDa fragment was not enhanced in cells. In addition, no corresponding to the size of rhIL-33 was detected in the or presence of we observed of full-length pro-IL-33 into culture supernatants specifically in THP-1 cells. secretion of mature IL-1β, as an of caspase-1 activation, was observed in and THP-1 cells we observed secretion of IL-33 in we investigated whether full-length pro-IL-33, like be to activate cell We thus mouse pro-IL-33 from transfected 293T total cell or using Although pro-IL-33 to it to using with bioactivity such as using a in a not of pro-IL-33 only be using as by in In fact, indicated that pro-IL-33 to protein in the of ST2-Fc not and this may thus for our in In addition, this observation that of pro-IL-33 to be to of pro-IL-33 to this of we used of pro-IL-33 to by acid as a to protein in 30-kDa pro-IL-33 was the only form of IL-33 in of to P815 cells induced IL-6 and this was in the presence of not Consistently, pro-IL-33 induced IL-6 secretion in wild-type but not in knock-out further suggesting that pro-IL-33 T1/ST2-dependent biological effects in mast cells. The to was in wild-type and knock-out that is no in the of IL-6 secretion by in knock-out cells. In of the with the of pro-IL-33 from cell lysates, we used in vitro synthesized as an of pro-IL-33 to biological effects In vitro synthesized mouse pro-IL-33 induced IL-6 in P815 cells, and this was in the presence of The two of mouse pro-IL-33, D88A and induced IL-6 in P815 cells, and was by Finally, we investigated potential in situ processing of pro-IL-33 to in the culture medium of P815 cells. cells not as assessed by reverse transcription-PCR not but we after addition to the cultures, pro-IL-33 be by other secreted or cell Indeed, we processing of pro-IL-33 to in and 18-kDa after a incubation with P815 cells, the of full-length pro-IL-33 and was between and data not The presence of was observed P815 cells were with the D88A and D106A of pro-IL-33 not It thus remains to be determined which of IL-33 indeed to In this study, we generation of a 20-kDa fragment of IL-33 in cell lysates, which is enhanced by incubation with caspase-1 in The of this fragment is than that of IL-33. We further that generation of this fragment is not by which not pro-IL-33 in an in vitro of In addition, our results indicate that caspase-1 cleavage is not required for pro-IL-33 secretion and bioactivity. Although it is that the IL-33 processing is to that of IL-1β, our data thus major differences between two We that the 20-kDa form of IL-33, which we observed using the mouse and human in corresponds to the mature form of IL-33 described by (1.Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. Gorman D.M. Bazan J.F. Kastelein R.A. Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2968) Google Scholar) after incubation of human pro-IL-33 with caspase-1. In this the of this IL-33 form was indeed not with that of data further that the cleavage reported in this was not by but rather was by which known to be present in V. S. C. A. F. J. Cell 2007; PubMed Scopus Google Scholar). Caspase-1 has indeed been observed previously to activation of other or M. Kanneganti T.D. Van Damme P. Vanden Berghe T. Vanoverberghe I. Vandekerckhove J. Vandenabeele P. Gevaert K. Núñez G. Mol. Cell. Proteomics. 2008; 7: 2350-2363Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, T. S. Biophys. 2005; PubMed Scopus Google Scholar). In of this we observed the presence of the 20-kDa form of IL-33 to a extent in of 293T cells, which not endogenous C. and G. before addition of recombinant caspase-1. generation of this fragment was observed with the D88A and D106A of mouse pro-IL-33, that it was of the presence of the two potential caspase-1 cleavage present in the N-terminal moiety of mouse Finally, our the of a conserved region corresponding to a potential caspase-1 cleavage site between species. generation of this 20-kDa form of IL-33 was observed not only in but in THP-1 cells. The as as to be observation is the of full-length pro-IL-33 into the supernatants of THP-1 cells, suggesting that pro-IL-33 can be secreted in the of caspase-1 with our the presence of full-length pro-IL-33 in the supernatants of activated THP-1 cells has been detected previously (9.Li H. Willingham S.B. Ting J.P. Re F. J. Immunol. 2008; 181: 17-21Crossref PubMed Scopus (33) Google Scholar). In this two of IL-33 were observed in THP-1 supernatants. We from our data that of IL-33 may be released into THP-1 supernatants at the of our Western blot full-length pro-IL-33 be further THP-1 culture supernatants by or cell as is the with P815 cells. The of pro-IL-33 by cells suggests pro-IL-33 is not a substrate for secretion may be dependent on caspase-1 observation is with a secretion of a number of which not caspase-1 M. A. S. Cell. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition to by activated cells, pro-IL-33 was recently to as an upon from endothelial or cells, which form C. Ortega N. Girard J.P. PLoS ONE. 2008; 3: e3331Crossref PubMed Scopus (939) Google Scholar). Finally, incubation with full-length pro-IL-33 induced T1/ST2-dependent biological effects in mast cells. However, we observed of processing of wild-type and pro-IL-33 in P815 culture medium the used for it is not clear at this whether the full-length form itself one or cleavage products the bioactive species. in to the cell we P815 culture medium an 18-kDa form of IL-33, which was in size to the recombinant In any P815 cells not that IL-33 processing is our data clearly in to IL-1β, IL-33 bioactivity not activation by caspase-1 cleavage. In conclusion, we the generation of a 20-kDa fragment of and which is from fragment is not produced by direct caspase-1 cleavage. In fact, our results indicate that pro-IL-33 is not a substrate for this enzyme. We observed that pro-IL-33 is released in full-length form by activated THP-1 cells and that addition of pro-IL-33 T1/ST2-dependent biological effects in mast cells. our results clearly indicate that caspase-1 cleavage is not required for IL-33 secretion and bioactivity. We for with lentiviral McKenzie for knock-out Donzé for rmIL-33 and and for and for with and for