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IL-33, the IL-1-Like Cytokine Ligand for ST2 Receptor, Is a Chromatin-Associated Nuclear Factor in Vivo

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IL-33, the IL-1-Like Cytokine Ligand for ST2 Receptor, Is a Chromatin-Associated Nuclear Factor in Vivo IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo Virginie Carriere*, Lucie Roussel*, Nathalie Ortega*, Delphine-Armelle Lacorre*, Laure Americh†, Luc Aguilar†, Ge´ rard Bouche*, and Jean-Philippe Girard*‡ *Laboratoire de Biologie Vasculaire, Equipe Labellise´e ‘‘La Ligue 2006,’’ Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique, Unite´Mixte de Recherche 5089, 205 Route de Narbonne, 31077 Toulouse, France; and †Endocube, Prologue Biotech, BP700, Rue Pierre et Marie Curie, 31319 Labe`ge Cedex, France Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved November 6, 2006 (received for review August 8, 2006) Recent studies indicate that IL-1␣ functions intracellularly in path- synthesized as a 31-kDa protein that lacks a clear signal peptide (3). ways independent of its cell surface receptors by translocating to This IL-33 precursor has been shown to be cleaved by caspase-1 in the nucleus and regulating transcription. Similarly, the chromatin- vitro, and it has been proposed that, similarly to IL-1␤ and IL-18 (1, associated protein HMGB1 acts as both a nuclear factor and a 2), IL-33 may require processing by caspase-1 for optimal biological secreted proinflammatory cytokine. Here, we show that IL-33, an activity (3). However, it remains to be determined whether IL-33 is IL-1-like cytokine that signals via the IL-1 receptor-related protein processed to a mature active form by caspase-1 in vivo. ST2 and induces T helper type 2-associated cytokines, is an endo- The observation that the precursor form of each member of the thelium-derived, chromatin-associated nuclear factor with tran- IL-1 family (8), with the exception of the IL-1 receptor antagonist scriptional repressor properties. We found that IL-33 is identical to IL-1Ra, lacks a signal peptide suggested persistence of an early NF-HEV, a nuclear factor preferentially expressed in high endothe- evolutionary role of these proteins as intracellular factors (9). lial venules (HEV), that we previously characterized. Accordingly, in Indeed, there is growing evidence for intracellular roles of cytokines situ hybridization demonstrated that endothelial cells constitute a and growth factors of the IL-1/FGF family. For instance, IL-1␣, major source of IL-33 mRNA in chronically inflamed tissues from which is rarely found in the extracellular compartment but rather is patients with rheumatoid arthritis and Crohn’s disease. Immuno- primarily a cell-associated cytokine (1, 10), has been proposed to staining with three distinct antisera, directed against the N-termi- regulate cell migration, proliferation, senescence, and differentia- nal part and IL-1-like C-terminal domain, revealed that IL-33 is a tion through intracrine mechanisms and intracellular pathways heterochromatin-associated nuclear factor in HEV endothelial cells independent of its cell-surface membrane receptors (9, 11–16). in vivo. Association of IL-33 with heterochromatin was also ob- Nuclear translocation of the IL-1␣ precursor, mediated by a con- served in human and mouse cells under living conditions. In sensus nuclear localization sequence (NLS) within its N-terminal addition, colocalization of IL-33 with mitotic chromatin was noted. (Nter) part (17), has been shown to be critical for the intracellular Nuclear localization, heterochromatin-association, and targeting functions of IL-1␣ (12, 13). Several nuclear targets of the IL-1␣ to mitotic chromosomes were all found to be mediated by an precursor have been identified that interact specifically with the evolutionarily conserved homeodomain-like helix–turn–helix mo- acidic Nter propiece but not the C-terminal (Cter) mature form (15, tif within the IL-33 N-terminal part. Finally, IL-33 was found to 16). These targets include the histone acetyltransferases p300, possess transcriptional repressor properties, associated with the PCAF and Gcn5 (16), and the growth suppressor necdin (15). The homeodomain-like helix–turn–helix motif. Together, these data identification of the histone acetyltransferases as nuclear partners suggest that, similarly to IL1␣ and HMGB1, IL-33 is a dual function of IL-1␣ is in agreement with a recent report demonstrating an protein that may function as both a proinflammatory cytokine and important role of the IL-1␣ precursor as an intracrine proinflam- an intracellular nuclear factor with transcriptional regulatory matory activator of transcription (9). Together, these observations properties. indicate that IL-1␣ is a dual-function protein that acts as both a nuclear factor and a proinflammatory cytokine (9). Interestingly, a heterochromatin ͉ nucleus ͉ endothelium ͉ HMGB1 similar duality of function has been shown for high-mobility group box 1 (HMGB1) protein, an abundant chromatin-associated pro- ytokines of the IL-1 family play a major role in a wide range tein involved in transcriptional regulation that is released by ne- Cof inflammatory, infectious, and autoimmune diseases (1). crotic cells and secreted by activated macrophages during inflam- The three best known members of this family, IL-1␣, IL-1␤, and mation and functions extracellularly as a potent proinflammatory IL-18, are highly inflammatory cytokines, and dysregulation of cytokine (18–20). their production or activity can lead to severe pathological events In this study, we show that IL-33, which we found to be identical (1, 2). IL-33 is the most recent addition to the IL-1 family (3). to the NF-HEV protein we characterized (21), is a nuclear factor IL-33 has been shown to induce T helper (Th) type 2 responses associated with heterochromatin in vivo and mitotic chromosomes (3) by signaling through the IL-1 receptor-related protein ST2 (IL-1 R4), an orphan member of the IL-1 receptor family (4, 5). Author contributions: V.C. and L.R. contributed equally to this work; J.-P.G. designed Treatment of mice with recombinant IL-33 resulted in blood research; V.C., L.R., N.O., D.-A.L., L. Americh, and G.B. performed research; V.C., L.R., L. eosinophilia, splenomegaly, and increased serum levels of IgE, Aguilar, and J.-P.G. analyzed data; and J.-P.G. wrote the paper. IgA, IL-5, and IL-13 (3). Exposure to IL-33 also caused severe The authors declare no conflict of interest. pathological changes in the lungs and gastrointestinal tract, This article is a PNAS direct submission. including eosinophilic and mononuclear infiltrates, increased Abbreviations: Cter, C-terminal; EC, endothelial cell; HEV, high endothelial venules; HTH, mucus production, and epithelial cell hyperplasia and hypertro- helix–turn–helix; ISH, in situ hybridization; IHC, immunohistochemistry; NLS, nuclear lo- phy (3). calization sequence; Nter, N-terminal; RA, rheumatoid arthritis. IL-33 shares with the other members of the IL-1 family a single ‡To whom correspondence should be addressed. E-mail: [email protected]. structural domain formed from 12 ␤ strands, arranged in a so-called This article contains supporting information online at www.pnas.org/cgi/content/full/ IL-1/FGF ␤-trefoil fold (6, 7). Similarly to IL-1␣ (271 aa, 30.6 kDa) 0606854104/DC1. and IL-1␤ (269 aa, 30.7 kDa) (1), IL-33 (270 aa, 30.7 kDa) is © 2006 by The National Academy of Sciences of the USA 282–287 ͉ PNAS ͉ January 2, 2007 ͉ vol. 104 ͉ no. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606854104 Downloaded by guest on September 26, 2021 Fig. 2. IL-33 is a heterochromatin-associated nuclear factor in HEV ECs in vivo.(A, D, and G) Double staining of human tonsils sections with HEV-specific mAb MECA79 and three distinct IL-33 antisera against the Nter and IL-1-like MEDICAL SCIENCES Fig. 1. Expression of IL-33 mRNA in ECs from chronically inflamed human Cter domains, preabsorbed with a control peptide, demonstrated abundant tissues. (A–C) ISH analysis of IL-33 mRNA in human tonsils (A), Crohn’s disease expression of endogenous IL-33 in the nuclei of HEV ECs in vivo. Nuclei were intestine (B), and RA synovium (C). Cryosections were hybridized with IL-33 counterstained with DAPI. (B, E, and H) Nuclear staining of HEV ECs with the sense and antisense riboprobes. ISH signals were detected with FITC- three IL-33 antisera was abrogated by preincubation of the antibodies with conjugated anti-biotin antibody (A and C) or incubation with Fast-red (B). In their corresponding IL-33 peptides. (C, F, and I) Higher magnification revealed C, ISH (green signal) was combined with IHC for the postcapillary venule that endogenous IL-33 accumulates in nuclear domains that colocalize with marker DARC (red signal), and nuclei were counterstained with DAPI (blue dense regions of DAPI staining, indicating association with heterochromatin. signal). (C Inset) Abundant IL-33 signal (green dots) in a DARC-positive post- capillary venule (red signal). (D) Detection of IL-33 mRNA in ECs freshly purified from human tonsils, Crohn’s disease intestine, and RA synovium by from human tonsils (Fig. 1A), Crohn’s disease intestine (Fig. 1B), using semiquantitative RT-PCR. HeLa epithelial cell line and GAPDH primers and RA synovium (Fig. 1C), whereas the sense probe gave only were used as controls. background signals. Combined ISH for IL-33 and immunohisto- chemistry (IHC) for postcapillary venule EC marker DARC re- in living cells, that possesses potent transcriptional-repressor prop- vealed expression of IL-33 mRNA in both DARC-positive and erties. Our findings suggest that IL-33, similarly to IL-1␣, may -negative ECs (Fig. 1C). Semiquantitative RT-PCR confirmed function as both a proinflammatory cytokine and an intracellular abundant expression of IL-33 mRNA in ECs freshly isolated from nuclear factor involved in transcriptional regulation. human tonsils, RA synovium, and Crohn’s intestine (Fig. 1D). In contrast, IL-33 mRNA was not detected in HeLa epithelial cancer Results cells.
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