Abstract: The interleukin-1 (IL-1)-like cytokine IL-33 is widely assumed to undergo proteolytic maturation by caspase-1. In this issue of Immunity, Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar show that IL-33 is not a caspase-1 substrate. IL-33 is inactivated by caspase-3 and -7 to prevent an inappropriate immune response during apoptosis, but not in necrosis. The interleukin-1 (IL-1)-like cytokine IL-33 is widely assumed to undergo proteolytic maturation by caspase-1. In this issue of Immunity, Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar show that IL-33 is not a caspase-1 substrate. IL-33 is inactivated by caspase-3 and -7 to prevent an inappropriate immune response during apoptosis, but not in necrosis. Interleukin-33 (IL-33) was initially described as "nuclear factor from high endothelial venules" (NF-HEV) because it resided in the nucleus of high endothelial cells (Baekkevold et al., 2003Baekkevold E.S. Roussigne M. Yamanaka T. Johansen F.E. Jahnsen F.L. Amalric F. Brandtzaeg P. Erard M. Haraldsen G. Girard J.P. Am. J. Pathol. 2003; 163: 69-79Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). These cells form "high endothelial venules" (HEVs) in secondary lymphoid tissue and serve as entry sites for extravasating lymphocytes. More recently, nuclear IL-33 was also detected in epithelial and non-HEV endothelial cells (Moussion et al., 2008Moussion C. Ortega N. Girard J.P. PLoS ONE. 2008; 3: e3331Crossref PubMed Scopus (901) Google Scholar). Its nuclear localization in these cells was explained by the presence of an amino-terminal homeodomain-like DNA-binding motif (Baekkevold et al., 2003Baekkevold E.S. Roussigne M. Yamanaka T. Johansen F.E. Jahnsen F.L. Amalric F. Brandtzaeg P. Erard M. Haraldsen G. Girard J.P. Am. J. Pathol. 2003; 163: 69-79Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar). Although the exact function of endogenous IL-33 in the nucleus has not been established, overexpression studies suggested a role in transcriptional repression (Carriere et al., 2007Carriere V. Roussel L. Ortega N. Lacorre D.A. Americh L. Aguilar L. Bouche G. Girard J.P. Proc. Natl. Acad. Sci. USA. 2007; 104: 282-287Crossref PubMed Scopus (783) Google Scholar). In 2005, mining of genomic and expressed sequence tag (EST) databases led to the rediscovery of IL-33 as an IL-1-like protein (Schmitz et al., 2005Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. et al.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2829) Google Scholar). It shares with other IL-1 family members the presence of a β-trefoil fold in its carboxy-terminus. Moreover, IL-33 was identified as an extracellular ligand for the orphan IL-1 receptor (IL-1R) family member ST2 (Schmitz et al., 2005Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. et al.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2829) Google Scholar), which signals in conjunction with IL-1R accessory protein (IL-1RAcP) (Ali et al., 2007Ali S. Huber M. Kollewe C. Bischoff S.C. Falk W. Martin M.U. Proc. Natl. Acad. Sci. USA. 2007; 104: 18660-18665Crossref PubMed Scopus (274) Google Scholar, Chackerian et al., 2007Chackerian A.A. Oldham E.R. Murphy E.E. Schmitz J. Pflanz S. Kastelein R.A. J. Immunol. 2007; 179: 2551-2555PubMed Google Scholar). IL-33 binding of the ST2 receptor on mast cells and T helper 2 (Th2) lymphocytes triggers secretion of proinflammatory and the Th2 cell-associated cytokines IL-4, IL-5, and IL-13 (Ali et al., 2007Ali S. Huber M. Kollewe C. Bischoff S.C. Falk W. Martin M.U. Proc. Natl. Acad. Sci. USA. 2007; 104: 18660-18665Crossref PubMed Scopus (274) Google Scholar, Chackerian et al., 2007Chackerian A.A. Oldham E.R. Murphy E.E. Schmitz J. Pflanz S. Kastelein R.A. J. Immunol. 2007; 179: 2551-2555PubMed Google Scholar, Schmitz et al., 2005Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. et al.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2829) Google Scholar). These findings paved the way to unraveling the role of IL-33 in inflammatory, infectious, and autoimmune diseases, but also raised the question how the protein migrates from its nuclear localization in endothelial and epithelial cells to the extracellular ST2 receptor on mast cells and other immune cells. A widely held explanation for the release of IL-33 is based on the unique secretory mechanism of two other cytokines of the IL-1 family, IL-1β and IL-18. Whereas most cytokines traffic through the Golgi complex prior to exocytosis, IL-1β and IL-18 are produced as biologically inactive precursors that reside in the cytosolic compartment. In response to an inflammatory insult, they are proteolytically matured by the cysteine protease caspase-1 (Lamkanfi and Dixit, 2009Lamkanfi M. Dixit V.M. Immunol. Rev. 2009; 227: 95-105Crossref PubMed Scopus (287) Google Scholar). Because of its homology to IL-1β and IL-18 and the absence of an apparent secretory signal peptide, IL-33 was widely assumed to be synthesized as a 31-kDa precursor from which the biologically active cytokine was liberated by caspase-1. In this issue of Immunity, Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar now showed this model to be incorrect. They show that concentrations of recombinant caspase-1 that achieve robust maturation of IL-1β fail to process human and mouse IL-33. In addition, full-length IL-33 displays potent biological activity, further arguing that unlike IL-1β and IL-18 precursors, IL-33 does not require caspase-1 processing. Another recently published report independently arrived at these conclusions (Talabot-Ayer et al., 2009Talabot-Ayer D. Lamacchia C. Gabay C. Palmer G. J. Biol. Chem. 2009; (in press. Published online May 22, 2009)https://doi.org/10.1074/jbc.M901744200Crossref PubMed Scopus (204) Google Scholar). A third concomitant report observed caspase-1-dependent processing of IL-33 in rabbit reticulocyte lysates; however, this led to inactivation of IL-33 rather than its activation (Cayrol and Girard, 2009Cayrol C. Girard J.P. Proc. Natl. Acad. Sci. USA. 2009; 106: 9021-9026Crossref PubMed Scopus (541) Google Scholar). Importantly, the identified cleavage site (DGVD178 in human IL-33 and DGVD175 in mouse IL-33) does not resemble preferred caspase-1 cleavage sites but closely matches processing sites of the apoptotic caspase-3 and -7. This suggests that recombinant caspase-1 may have activated endogenous caspase-3 or -7 in the reticulocyte lysates. Indeed, recombinant caspases-3 and -7 readily processed human and mouse IL-33 after Asp178 and Asp175, respectively (Cayrol and Girard, 2009Cayrol C. Girard J.P. Proc. Natl. Acad. Sci. USA. 2009; 106: 9021-9026Crossref PubMed Scopus (541) Google Scholar, Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). Further, unlike IL-33D178A mutant, wild-type IL-33 was processed in cells undergoing apoptosis in response to a variety of DNA-damaging agents and when treated with anti-Fas, TRAIL, or TNF-α plus cycloheximide (Cayrol and Girard, 2009Cayrol C. Girard J.P. Proc. Natl. Acad. Sci. USA. 2009; 106: 9021-9026Crossref PubMed Scopus (541) Google Scholar, Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). Finally, IL-33 was cleaved in apoptotic murine embryonic fibroblast (MEF) cells lacking caspase-1 or -7, but not those deficient for caspase-3 (Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). Surprisingly, although caspase-7-cleaved IL-33 failed to activate the ST2 receptor, its binding to the receptor was comparable to that of full-length IL-33 (Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). However, this experiment has to be interpreted with caution because the caspase-7-incubation setup was not devoid of unprocessed IL-33. It would be interesting to assess receptor binding of the separately generated N- and C-terminal (IL-331–178 and IL-33179–270) fragments, although it is clear that they are incapable of activating ST2 receptor signaling (Cayrol and Girard, 2009Cayrol C. Girard J.P. Proc. Natl. Acad. Sci. USA. 2009; 106: 9021-9026Crossref PubMed Scopus (541) Google Scholar, Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). Strong ST2 binding of one of the fragments in the absence of receptor activation may suggest a potentially novel approach to interfere with ST2 signaling. An increased vulnerability to degradation by serum proteases was suggested as a potential explanation for the reduced biological activity of caspase-7-cleaved IL-33 in the absence of a loss of receptor binding (Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). Because Asp178 is located within the IL-1-like β-trefoil domain of IL-33 (Schmitz et al., 2005Schmitz J. Owyang A. Oldham E. Song Y. Murphy E. McClanahan T.K. Zurawski G. Moshrefi M. Qin J. Li X. et al.Immunity. 2005; 23: 479-490Abstract Full Text Full Text PDF PubMed Scopus (2829) Google Scholar), processing at this position may also destabilize the β-trefoil structure and prevent crucial activating interactions with the ST2 receptor. But when is IL-33 secreted? Unlike IL-1β, which is readily secreted by macrophages and dendritic cells after stimulation with LPS and PMA, IL-33 remained intracellular (Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). In apoptotic macrophages, caspase-mediated processing ensured inactivation of IL-33, but the processing fragments were nevertheless kept cell associated. In contrast, most IL-33 was released from macrophages induced to undergo necrotic cell death (Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). As expected, IL-33 was not processed in necrotic endothelial cells (Cayrol and Girard, 2009Cayrol C. Girard J.P. Proc. Natl. Acad. Sci. USA. 2009; 106: 9021-9026Crossref PubMed Scopus (541) Google Scholar) because caspases are not activated during this cell death process. Together, these findings suggest that IL-33 is specifically released during necrotic cell death, which is thought to be associated with tissue damage during trauma or infection. Under these conditions, extracellular IL-33 may engage the ST2 receptor on mast cells and other immune cells in order to alert the immune system of tissue damage and infection and to promote the initiation of healing responses (Figure 1). In support of this hypothesis, IL-33 is highly expressed in endothelial cells of most organs and in the epidermal and gastrointestinal epithelium (Moussion et al., 2008Moussion C. Ortega N. Girard J.P. PLoS ONE. 2008; 3: e3331Crossref PubMed Scopus (901) Google Scholar). These tissues may become exposed to pathogens, allergens, and other environmental agents that can trigger tissue damage. In this respect, IL-33 appears highly reminiscent of IL-1α and HMGB1, two dual-function proteins that play important roles as both intracellular nuclear proteins and extracellular cytokines. Moreover, all three proteins lack classical secretion signals and display cytokine activity independently of processing. In addition, all three are released by necrotic cells, but kept intracellular during apoptosis. Because of these features, HMGB1 and IL-1α have been referred to as "endogenous danger signals" or "alarmins." The work by Lüthi et al., 2009Lüthi A.U. Cullen S.P. McNeela E.A. Duriez P.J. Afonina I.S. Sheridan C. Brumatti G. Taylor R.C. Kersse K. Vandenabeele P. et al.Immunity. 2009; 31 (this issue): 84-98Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar now also bestows IL-33 with this title. M.L. and V.M.D. are employees of Genentech, Inc. Suppression of Interleukin-33 Bioactivity through Proteolysis by Apoptotic CaspasesLüthi et al.ImmunityJune 25, 2009In BriefInterleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent proteolysis, similar to the proinflammatory cytokines IL-1β and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-κB transcription factor. Full-Text PDF Open Archive