Cellular & Molecular Immunology (2010) 7, 260–262 ß 2010 CSI and USTC. All rights reserved 1672-7681/10 $32.00 www.nature.com/cmi

MINI REVIEW

The enigmatic processing and secretion of -33

Weihua Zhao and Zhiqing Hu

Interleukin-33 (IL-33) is the most attractive novel identified as an IL-1 family member. IL-33 was first named NF-HEV (nuclear factor from high endothelial venules), as it was known to interact with nuclear chromatin although its exact intracellular functions are still to be clarified. IL-33 is now recognized as the specific for the orphan IL-1 family member ST2 and to be involved in polarization of T cells towards T helper 2 phenotype and in activation of mast cells, bosophils, and natural killer cells. It is essential for IL-33 to be extracellularly released in order to bind to the ST2 receptor and consequently play a crucial role in inflammatory, infectious and autoimmune diseases. However, like the IL-1 family members, IL-1b and IL-18, IL-33 mRNA is translated without a signal sequence for secretion. Additionally, IL-33 cannot be released by the processing and secretion mechanism shared by IL-1b and IL-18 as IL-33 is not a substrate of caspase-1 and does not require proteolysis for activation. In contrast, IL-33 can be inactivated by apoptotic caspases. Accordingly, IL-33 is proposed to be released as an alarmin from necrotic cells but to be deleted during . Besides the known autocrine, paracrine, intracrine, juxtacrine and retrocrine mechanisms of cellular interaction with , release by necrotic cells is another pathway for a cytokine to display its function, which we suggest might be called ‘necrocrine’. This mini review summarizes recent progress of how IL-33 displays potential immunoregulatory roles with a particular focus on its enigmatic production. Cellular & Molecular Immunology (2010) 7, 260–262; doi:10.1038/cmi.2010.3; published online 22 March 2010

Keywords: Interleukin-33; Interleukin-1 family; Caspase-1; Intracrine; Necrocrine

INTRODUCTION hydrolyzing role of caspase-1 in the full-length IL-33 is also brought In the ever-growing list of cytokines, interleukin-33 (IL-33), one of the into question by recent findings.42–46 For novel insights into the bio- IL-1 family members (also called IL-1F11), is a most interesting novel logical functions of the IL-33/ST2 pathway, it is crucial to address such cytokine. This cytokine was first named in 2003 as NF-HEV (nuclear questions and identify possible mechanisms underlying these discrep- factor from high endothelial venules), as it was known to interact with ancies. This mini review summarizes the recent progress in our under- nuclear chromatin in an intracrine manner.1 It was rediscovered in standing of how IL-33 displays a potential immunoregulatory role 2005 as the specific extracellular ligand for the orphan IL-1 receptor with a particular focus on the mechanisms relating to its processing family member ST2, and named IL-33.2 ST2 (also known as IL-1RL1, and secretion. DER4, T1 and FIT-1) belongs to the Toll-like/IL-1 receptor superfam- ily.3–5 It has been well documented to be the cellular marker for differ- CAN IL-33 BE SECRETED? entiated T helper 2 (Th2) cells6,7 and to be expressed on mast cells.8,9 It is essential for IL-33 to be secreted or released in order to bind to the As a nuclear factor, the intracellular functions of IL-33 remain to be ST2 receptor although an intracrine mechanism has been reported.1,10 further clarified, although overexpression studies suggested a role as a However, as with the IL-1 family members IL-1b and IL-18, IL-33 transcriptional repressor.10 As an extracellular cytokine, binding of IL- lacks a classical signal sequence necessary for secretion via the endo- 33 to the ST2 receptor activates nuclear factor-kB and mitogen-acti- plasmic reticulum/Golgi pathway.2,11,33,42,43 Active caspase-1 has been vated kinases,3–5,11 and is involved in the polarization of T cells confirmed to be a regulator of the unconventional secretion of such towards the Th2 cell phenotype2,6,7,11–14 and in activation of mast .47 IL-1b and IL-18 are well known to be synthesized as a cells,15–20 bosophils,14,20–24 eosinophils,24–26 and natural killer biologically inactive 33-kDa pro-IL-1b and 23-kDa pro-IL-18 which cells.14,27 IL-33 must be present extracellularly in order to play the reside in the cytosolic compartment and to be subsequently cleaved by crucial role in inflammatory, infectious and autoimmune diseases caspase-1 to secrete the active 17-kDa IL-1b and 18-kDa IL-18.47,48 IL- including anaphylactic shock, asthma, rheumatoid arthritis, athero- 33 is also proposed to be synthesized as a 31-kDa pro-IL-33 and to be sclerosis, systemic sclerosis and cardiovascular diseases.5,11,28–38 hydrolyzed by caspase-1 to release an active 18-kDa IL-33 via the same However, complexities and a number of discrepancies in the proces- processing and secretion mechanism as IL-1b and IL-18.2,39–41 sing and secretion of IL-33 have been uncovered in the accumulated However, this conclusion is currently being questioned by controver- data. For example, full-length 31-kDa IL-331–270 has been reported, sial findings showing that the full-length 31-kDa IL-33 is active variously, as the immature/mature or inactive/active IL-33.2,39–46 The and that caspase-1 cleavage is not required for IL-33 activation and

Department of Microbiology and Immunology, Showa University School of Medicine, Tokyo, Japan Correspondence: Dr ZQ Hu, Department of Microbiology and Immunology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. E-mail: [email protected] Received 3 December 2009; Revised 28 December 2009; accepted 17 January 2010 The novel cytokine interleukin-33 WH Zhao and ZQ Hu 261

42–46 secretion. The so-called pro-IL-331–270 does not possess a definite IL-33 is an extremely complex process. Further efforts are needed to caspase-1 cleavage site and caspase-1 does not cleave pro-IL-33 at the reach a conclusion, though addressing the following issues should help proposed site in vivo. The 18-kDa IL-33112–270 that has been used as a clarify the situation. soluble recombinant cytokine is an artificially truncated form of IL- First, different cell sources may result in different and even contra- 42–44 331–270 and not a product of caspase-1 hydrolyzation. In contrast, dictory conclusions. As the number of publications using recombinant the two caspase-1 cleavage products, IL-331–178 and IL-33179–270,do IL-33 grows, the functional properties of IL-33 are becoming well not activate ST2 and the processing by caspase-1 results in attenuation characterized. However, the cellular sources and stimulants for either or inactivation of IL-33.42–45 Instead of caspase-1, calpain, a calcium- up- or downregulation of IL-33 expression are still largely unknown. dependent cysteine protease, has been reported to mediate the full- IL-33 is reported to be constitutively expressed by endothelial,1,2,62,63 length IL-33 processing in vivo.49 However, a recent report shows that epithelial63 and smooth muscle cells.2,34 Additionally, inducible IL-33 can be normally released from caspase-1-deficient, caspase-8 expression of IL-33 has been described in activated ,2,46 inhibitor-treated and calpain inhibitor-treated murine macro- and exposed to tumour-necrosis factor-a and phages.46 These observations, together with the fact that the full-length IL-1b,2 astrocytes stimulated by lipopolysaccharide and adenosine 31-kDa IL-33 is active and is not a substrate of caspase-1, indicate the triphosphate,61 and activated by tumour-necrosis factor- major differences between IL-1b/IL-18 and IL-33. a.64 IL-33 is also induced when cultured endothelial cells reach The question, then, is, how is IL-33 secreted or released? Several confluence and stop proliferating.62 Therefore, upon stimulation, dif- groups have compared the similarities between IL-33 and another IL-1 ferent cell populations may secrete or release IL-33 through quite family member IL-1a.42,44 Like IL-33, IL-1a also displays dual actions different pathways. as both an intracellular nuclear factor and an extracellular cyto- Second, the limitation of in vitro or in vivo system and the gap kine.50,51 IL-1a precursor (pro-IL-1a) is also biologically active and between in vitro and in vivo experiments cannot be ignored when data capable of binding to the IL-1 receptor.52 The pro-IL-1a is not a are interpreted. While in vitro studies are indispensable, particularly in substrate of caspase-1 but can be cleaved by calpain to release 17- cytokine research using cultured cells and various stimulants, it is kDa IL-1a.11,53–55 As with an alarmin such as high mobility group essential to avoid the overinterpretation that often accompanies pre- protein-1, release of IL-1a by necrotic cells is supposed to be the major dictions of the significance of an in vitro finding. Some of the findings route of its production.33,42–44,56,57 Therefore, IL-33 release might observed in vitro may not occur in vivo. Therefore, conclusions drawn resemble that of pro-IL-1a and high mobility group protein-1. from in vitro studies should be restricted to in vitro settings until they Lu¨thi et al. have confirmed that IL-33 is not a substrate of inflammat- are confirmed in an in vivo model system. ory caspase-1, -4 and -5 and can be released by necrotic cells without In summary, a thorough understanding of the complexity of IL-33 being inactivated. In contrast, IL-33 can be efficiently cleaved by cas- processing and secretion is essential to further define the biological pase-3 and -7 at physiological concentrations within apoptotic cells, roles and the contributions of IL-33 in various inflammatory, infec- 42,43 resulting in reduction of the proinflammatory activity of IL-33. tious and autoimmune diseases. Cayrol et al. have also confirmed that the full-length IL-33, which is constitutively expressed by endothelial cells in most normal human tissues, can be released into the extracellular space after endothelial cell 44 damage or mechanical injury. Accordingly, IL-33 is proposed to be 1 Baekkevold ES, Roussigne´M, Yamanaka T, Johansen FE, Jahnsen FL, Amalric F et al. released from necrotic cells as one of the alarmins but to be deleted by Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in 42–44 human high endothelial venules. Am J Pathol 2003; 163:69–79. endogenous apoptotic caspases in cells undergoing apoptosis. 2 Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK et al. IL-33, an Besides the known autocrine, paracrine, intracrine, juxtacrine and interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and retrocrine mechanisms of cellular interaction with cytokines, the induces T helper type 2-associated cytokines. 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