IL-33, the IL-1-like cytokine ligand for ST2 receptor, is a chromatin-associated nuclear factor in vivo

Abstract

Recent studies indicate that IL-1alpha functions intracellularly in pathways independent of its cell surface receptors by translocating to the nucleus and regulating transcription. Similarly, the chromatin-associated protein HMGB1 acts as both a nuclear factor and a secreted proinflammatory cytokine. Here, we show that IL-33, an IL-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines, is an endothelium-derived, chromatin-associated nuclear factor with transcriptional repressor properties. We found that IL-33 is identical to NF-HEV, a nuclear factor preferentially expressed in high endothelial venules (HEV), that we previously characterized. Accordingly, in situ hybridization demonstrated that endothelial cells constitute a major source of IL-33 mRNA in chronically inflamed tissues from patients with rheumatoid arthritis and Crohn's disease. Immunostaining with three distinct antisera, directed against the N-terminal part and IL-1-like C-terminal domain, revealed that IL-33 is a heterochromatin-associated nuclear factor in HEV endothelial cells in vivo. Association of IL-33 with heterochromatin was also observed in human and mouse cells under living conditions. In addition, colocalization of IL-33 with mitotic chromatin was noted. Nuclear localization, heterochromatin-association, and targeting to mitotic chromosomes were all found to be mediated by an evolutionarily conserved homeodomain-like helix-turn-helix motif within the IL-33 N-terminal part. Finally, IL-33 was found to possess transcriptional repressor properties, associated with the homeodomain-like helix-turn-helix motif. Together, these data suggest that, similarly to IL1alpha and HMGB1, IL-33 is a dual function protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties.

Fig. 1.

Expression of IL-33 mRNA in ECs from chronically inflamed human tissues. (A–C) ISH analysis of IL-33 mRNA in human tonsils (A), Crohn's disease intestine (B), and RA synovium (C). Cryosections were hybridized with IL-33 sense and antisense riboprobes. ISH signals were detected with FITC-conjugated anti-biotin antibody (A and C) or incubation with Fast-red (B). In C, ISH (green signal) was combined with IHC for the postcapillary venule marker DARC (red signal), and nuclei were counterstained with DAPI (blue signal). (C Inset) Abundant IL-33 signal (green dots) in a DARC-positive postcapillary venule (red signal). (D) Detection of IL-33 mRNA in ECs freshly purified from human tonsils, Crohn's disease intestine, and RA synovium by using semiquantitative RT-PCR. HeLa epithelial cell line and GAPDH primers were used as controls.

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 Cter domains, preabsorbed with a control peptide, demonstrated abundant expression of endogenous IL-33 in the nuclei of HEV ECs in vivo. Nuclei were counterstained with DAPI. (B, E, and H) Nuclear staining of HEV ECs with the three IL-33 antisera was abrogated by preincubation of the antibodies with their corresponding IL-33 peptides. (C, F, and I) Higher magnification revealed that endogenous IL-33 accumulates in nuclear domains that colocalize with dense regions of DAPI staining, indicating association with heterochromatin.

Fig. 3.

IL-33 associates with heterochromatin and mitotic chromatin in living cells. (A–C) Association of IL-33-GFP protein with heterochromatin was observed under living conditions both in human HEK-293T epithelial cells (A) and mouse 3T3 fibroblasts (B). Colocalization of IL-33-GFP with dense regions of Hoechst staining was found at the perinucleolar heterochromatin in human cells and the pericentromeric heterochromatin in mouse cells (A and B, arrowheads), whereas GFP alone localized throughout the cell (C). (D) Heterochromatin association of IL-33 in living cells was also observed with an Nter GFP tag. (E–G) During mitosis, IL-33-GFP was found associated with mitotic chromatin (F), whereas GFP alone was distributed throughout the whole cell (E). Association of IL-33-GFP with mitotic chromosomes was retained in fixed cells (G). Staining of mitotic chromosomes with IL-33 Nter antibody was blocked with the Nter peptide. (H) Western blot analysis of chromatin extracts with the three IL-33 antisera (Nter, Cter1, and Cter2) and control histone H3 antibody. Chromatin fractions were prepared by digestion of nuclei with DNase and extraction with 0.4 M NaCl and 0.8 M NaCl. Residual pellet corresponds to proteins not extracted at 0.8 M NaCl.

Fig. 4.

An evolutionarily conserved homeodomain-like HTH motif within the IL-33 Nter part is necessary and sufficient for nuclear localization, heterochromatin association, and targeting to mitotic chromosomes. (A) Diagram of IL-33 and its deletion- or point-mutant derivatives. The capacity of each mutant to accumulate in the nucleus and associate with heterochromatin and mitotic chromatin was determined. (B–E) The different IL-33 mutants fused to GFP were expressed in HEK-293T cells, and their subcellular localization in interphase (B–D) or mitotic (E) cells was analyzed by fluorescence microscopy. DNA was counterstained with Hoechst 33342 in living cells and with DAPI in fixed cells.

Fig. 5.

IL-33 has potent transcriptional repressor properties. (A and B) Human HEK-293T epithelial cells were transiently transfected with increasing amounts (10, 100, or 500 ng) of expression vectors for GAL4-DB fusions of IL-33 (Gal4-IL33) or Suv39H1 (Gal4-Suv), along with reporter vector pLex-Gal4 in which luciferase gene transcription is under the control of five GAL4-DB-binding sites (A) or with the same reporter vector deleted of the GAL4-DB-binding sites (B). IL-33 not fused to GAL4-DB (IL33) was also tested as a control. Luciferase activities were normalized by cotransfection with pRL-CMV plasmid and expressed relative to the values obtained with empty expression vector pCMV-2N3T (control). Results are means and standard deviations of three independent transfection experiments. (C) Fold repression was calculated by dividing the normalized luciferase activity of cells expressing GAL4-DB alone (Gal4) by the activity of the IL-33 (Gal4-IL33) and Suv39H1 (Gal4-Suv) fusion proteins (500-ng expression vector). (D) The IL-33 Nter part and homeodomain-like HTH motif, but not the IL-1-like Cter part, exhibit transcriptional repressor activity. IL-33 full-length (1–270) or deletion mutants (1–65, 1–111, and 112–270) were expressed as GAL4-DB fusions (10-, 100-, 500-, or 750-ng expression vectors) and tested for their ability to repress the pLex-Gal4 luciferase reporter. Normalized luciferase activities were determined as described in A.