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.2009 Jun 2;106(22):9021-6.
doi: 10.1073/pnas.0812690106. Epub 2009 May 13.

The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1

Affiliations

The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1

Corinne Cayrol et al. Proc Natl Acad Sci U S A..

Abstract

IL-33 is a chromatin-associated cytokine of the IL-1 family that has recently been linked to many diseases, including asthma, rheumatoid arthritis, atherosclerosis, and cardiovascular diseases. IL-33 signals through the IL-1 receptor-related protein ST2 and drives production of pro-inflammatory and T helper type 2-associated cytokines in mast cells, T helper type 2 lymphocytes, basophils, eosinophils, invariant natural killer T cells, and natural killer cells. It is currently believed that IL-33, like IL-1beta and IL-18, requires processing by caspase-1 to a mature form (IL-33(112-270)) for biological activity. Contrary to the current belief, we report here that full-length IL-33(1-270) is active and that processing by caspase-1 results in IL-33 inactivation, rather than activation. We show that full-length IL-33(1-270) binds and activates ST2, similarly to IL-33(112-270), and that cleavage by caspase-1 does not occur at the site initially proposed (Ser(111)), but rather after residue Asp(178) between the fourth and fifth predicted beta-strands of the IL-1-like domain. Surprisingly, the caspase-1 cleavage site (DGVD(178)G) is similar to the consensus site of cleavage by caspase-3, and IL-33 is also a substrate for this apoptotic caspase. Interestingly, we found that full-length IL-33, which is constitutively expressed to high levels by endothelial cells in most normal human tissues, can be released in the extracellular space after endothelial cell damage or mechanical injury. We speculate that IL-33 may function, similarly to the prototypical alarmins HMGB1 and IL-1alpha, as an endogenous danger signal to alert cells of the innate immune system of tissue damage during trauma or infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Processing of full-length IL-33 by caspase-1 generates a 20–22 kDa cleavage product that does not correspond to the IL-1-like domain. (A) Recombinant caspase-1 cleaves full-length IL-33 and pro-IL1β in vitro. Fluorescently labeled proteins were incubated for 2 h at 37 °C with increasing amounts of recombinant caspase-1 (0.05, 0.15, 0.5, or 1 unit) and then analyzed by SDS/PAGE and fluorography. Cleavage was abrogated by the caspase-1 inhibitor Ac-YVAD-CHO. (B) Recombinant caspase-3 cleaves full-length IL-33 in vitro. Fluorescently labeled IL-33 was incubated with increasing amounts of recombinant caspase-3 (0.05, 0.15, 0.5, or 1 unit) as described inA. Cleavage was abrogated by the caspase-3 inhibitor Ac-DEVD-CHO. (C) The 20–22 kDa caspase-1 cleavage product of IL-33 is recognized by IL-33 Nter antibodies but not by antibodies to the C terminus (Nessy-1, anti-myc). Fluorescently labeled or unlabeled IL-33 proteins, containing a C-terminal myc-epitope tag, were cleaved with 0.5 unit of caspase-1 as described inA and analyzed by fluorography (fluorescent IL-33) or Western blot (unlabeled IL-33) with IL-33 Nter, Nessy-1, or anti-myc antibodies.
Fig. 2.
Fig. 2.
Caspase-1 processing of IL-33 occurs after residue Asp178 within the IL-1-like domain. (A) Primary structure of human IL-33. The N-terminal domain involved in IL-33 nuclear activities and the IL-1-like domain, with its 12 predicted β-strands (black boxes), are indicated. The sequence surrounding the caspase-1 and caspase-3 cleavage site (Asp178) is shown for both human (Hs) and mouse (Mm) IL-33.CBM, chromatin-binding motif (aa 40–58). (B) An IL-331–178 deletion protein generated by in vitro translation (not tagged with myc-epitope) co-migrates on SDS/PAGE with the 20–22 kDa caspase-1 cleavage product of IL-33. Fluorescently labeled IL-33 protein was incubated with 0.5 units of caspase-1 for 2 h at 37 °C (with or without prior incubation with Ac-YVAD-CHO inhibitor) and analyzed by SDS/PAGE and fluorography. (C andD) Mutation of Asp178 to alanine abrogates cleavage of IL-33 by both caspase-1 (C) and caspase-3 (D). Fluorescently labeled IL-331–270 and IL-33D178A proteins were incubated with recombinant caspase-1 (C) or caspase-3 (D) as described inB. The Ac-YVAD-CHO and Ac-DEVD-CHO inhibitors were used at 100 μM. Asterisk indicates non-specific band. (E) Mutation of Asp178 to alanine abrogates cleavage of IL-33 by endogenous caspases during doxorubicin-induced apoptosis. U20S cells were transfected with IL-331–270 or IL-33D178A expression vectors and treated 24 h later with doxorubicin in the presence or absence of the pan-caspase inhibitor Z-VAD-fmk. Proteins were analyzed 24 h later by Western blot analysis with IL-33 mAb 305B. (F) Endogenous IL-33 in primary human endothelial cells (treated with control or IL-33 siRNA) was detected by Western blot analysis with IL-33 mAb 305B. (G) Endogenous IL-33 is cleaved by endogenous caspases in endothelial cells treated with the apoptosis-inducing agent staurosporine. Endothelial cells were treated with staurosporine in the presence or absence of the pan-caspase inhibitor Z-VAD-fmk. Proteins were analyzed by Western blot analysis with IL-33 mAb 305B or PARP mAb (used as a control). IL-331–270 and IL-331–178 proteins (not tagged with myc-epitope), generated by in vitro translation, are shown (Right).
Fig. 3.
Fig. 3.
Full-length IL-331–270 is able to bind and activate the ST2 receptor. (A) Pull-down of full-length IL-331–270 with ST2-Fc fusion protein. Full-length (IL-331–270), C-terminal IL-1-like domain (IL-33112–270), and N-terminal domain (IL-331–111) proteins tagged with myc-epitope at their C terminus were incubated with ST2-Fc for 16 h at 4 °C and precipitated with protein-G agarose beads. The precipitates were separated by SDS/PAGE and analyzed by Western blot with anti-myc antibody. Rabbit reticulocyte lysate (RRL) is an un-programmed lysate. (B) Pull-down of endogenous IL-33 with ST2-Fc fusion protein. Endothelial cell freeze-thaw extracts were incubated with ST2-Fc and the precipitates were analyzed by Western blot with IL-33 mAb 305B. Asterisk indicates non-specific band. (C andD) Full-length IL-331–270 activates an ST2-dependent NFκB-GFP reporter gene. Assays were performed in HEK293T cells transfected with plasmids pNF-κB-hrGFP and pEF-BOS-hST2, using in vitro translated IL-331–270, IL-33112–270, and IL-331–111 proteins, as described inMaterials and Methods. Cells were analyzed for GFP expression by fluorescence microscopy (C) and flow cytometry (D). The percentage increase in GFP+ cells is shown (Below). Results are shown as means and SDs of 3 independent transfection experiments.
Fig. 4.
Fig. 4.
The 2 caspase-1 cleavage products, IL-331–178 and IL-33179–270, are not able to activate ST2. (A andB) The capacity of IL-331–178 and IL-33179–270 to activate ST2-dependent signaling was analyzed using an ST2-dependent NFκB-GFP reporter gene. Assays were performed in HEK293T cells transfected with plasmids pNF-κB-hrGFP and pEF-BOS-hST2 using in vitro translated IL-331–270, IL-33179–270, and IL-331–178 proteins as described inMaterials and Methods. Cells were analyzed for GFP expression by fluorescence microscopy (A) and flow cytometry (B). The percentage increase in GFP+ cells is shown (Below). Results are shown as means and SDs of 3 independent transfection experiments. (C) The capacity of IL-33 and deletion mutants to activate the IL-33-responsive mast cell line MC/9 was analyzed by determining IL-6 levels in supernatants using an ELISA. Results are shown as means and SDs of 3 separate data points.
Fig. 5.
Fig. 5.
Full-length IL-33 is released by damaged endothelial cells. (A-D) Western blot analysis of confluent primary human endothelial cells lysates or supernatants was performed using antibodies against IL-33 (AT-110) and HMGB1. (A) Similar amounts of HMGB1 were observed in the presence or absence of siRNA to IL-33. (B) IL-33 and HMGB1 were released in the supernatants after scraping of the cells from the substratum (followed by 20 min incubation at 37 °C) or scratching the endothelial monolayer with a surgical scalpel. Supernatants were collected from wounded cells and the presence of IL-33 and HMGB1 was assayed in both pellets and supernatants concentrated by TCA precipitation or filtration on Vivaspin columns. Asterisk indicates non-specific band. (C) Higher amounts of IL-33 and HMGB1 were released in the supernatants after endothelial cell damage induced by repeated cycles of freezing and thawing. (D) IL-33 and HMGB1 were also released in the supernatants after treatment of the endothelial cells for 5 min at 37 °C with non-ionic detergents 0.2% Nonidet P-40 and 0.2% Triton X-100.
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