The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1 (original) (raw)

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-1β and IL-18, requires processing by caspase-1 to a mature form (IL-33112–270) for biological activity. Contrary to the current belief, we report here that full-length IL-331–270 is active and that processing by caspase-1 results in IL-33 inactivation, rather than activation. We show that full-length IL-331–270 binds and activates ST2, similarly to IL-33112–270, and that cleavage by caspase-1 does not occur at the site initially proposed (Ser111), but rather after residue Asp178 between the fourth and fifth predicted β-strands of the IL-1-like domain. Surprisingly, the caspase-1 cleavage site (DGVD178G) 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-1α, as an endogenous danger signal to alert cells of the innate immune system of tissue damage during trauma or infection.

Keywords: inflammation, Interleukin, endothelial cell

Cytokines of the IL-1 family play a major role in a wide range of inflammatory, infectious, and autoimmune diseases (1, 2). IL-33 [previously known as nuclear factor from high endothelial venule, or NF-HEV (3)] is the most recent addition to the IL-1 family (4, 5). Based on animal model studies and analyses of diseased tissues from patients, IL-33 has been proposed to represent a promising therapeutic target for several important diseases, including asthma and other allergic diseases (4), rheumatoid arthritis (5, 6), atherosclerosis (7), and cardiovascular diseases (8, 9). IL-33 has been shown to signal through the IL-1 receptor-related protein ST2 (4) and to drive production of cytokines [both pro-inflammatory and T helper type 2 (Th2)-associated cytokines] and chemokines in mast cells, Th2 lymphocytes, basophils, eosinophils, invariant natural killer (NK) T cells, and NK cells (4, 10–19). IL-33 signaling has also been shown to require the IL-1 receptor (IL-1R) accessory protein, IL-1RAcP, indicating that IL-33 shares with IL-1 not only structural homology but also signaling pathways (11, 14).

We initially discovered IL-33 as a nuclear factor abundantly expressed in endothelial cells of high endothelial venules in lymphoid organs (3, 5), but we (20) and others (21) have recently found that IL-33 is also constitutively expressed to high levels in the nucleus of endothelial cells in other human tissues. These observations indicated that IL-33 is widely expressed along the vascular tree and that endothelial cells constitute a major cellular source of IL-33 in most human tissues (20, 21). We also showed that nuclear IL-33 possesses transcriptional regulatory properties and associates with chromatin in vivo (5). Recently, we found that IL-33 mimics Kaposi sarcoma herpesvirus for attachment to chromatin, and docks, through a short chromatin-binding peptide, into the acidic pocket formed by the histone H2A-H2B dimer at the surface of the nucleosome (22). Together, our findings suggested IL-33 is a dual-function protein that may play important roles as both a cytokine and an intracellular nuclear factor (5, 22). A similar duality of function has previously been shown for IL-1α and chromatin-associated cytokine HMGB1. IL-1α, a cell-associated cytokine, exhibits potent pro-inflammatory cytokine activities mediated by the cell surface IL-1 receptors (1) but also functions intracellularly by translocating to the nucleus and regulating transcription (23). HMGB1 regulates transcription and chromatin structure in the nucleus (24) but also functions extracellularly as a cytokine when secreted by activated macrophages during inflammation (25) or released by necrotic cells (26). IL-1α has also been shown to be released by dying cells (1, 27–29), and HMGB1 and IL-1α were thus defined as endogenous “danger signals” or “alarmins” that may alert the immune system after tissue damage during trauma or infection (30).

It is currently believed that IL-33, like classical IL-1 family members IL-1β and IL-18, requires maturation by caspase-1 for optimal biological activity. This is based on the initial report by Schmitz et al., who indicated caspase-1 cleaves IL-33 after residue Ser111, resulting in the production of mature IL-33112–270 (4). Most of the studies that have been published to date used a recombinant IL-33 protein corresponding to this mature form. Here, we show that full-length IL-331–270 is biologically active, and that cleavage by caspase-1 does not occur at the site initially proposed but rather after residue Asp178 within the IL-1-like domain. Consequently, caspase-1 processing results in inactivation of IL-33, rather than activation. We also demonstrate that full-length IL-331–270 can be released in the extracellular space after endothelial cell damage or injury. We discuss the possibility that IL-33 may function as an endogenous danger signal, like the prototypical alarmins IL-1α and HMGB1.

Results

The 20–22 kDa Caspase-1 Cleavage Product of IL-33 Does Not Correspond to the IL-1-Like Domain.

As previously reported by Schmitz et al. (4), we confirmed that in vitro translated human IL-33 can be cleaved by caspase-1 to a 20–22 kDa form, as determined by SDS/PAGE (Fig. 1A). As expected, pro-IL-1β was also cleaved by caspase-1 to the IL-1β mature form. Cleavage was abrogated in both cases by the caspase-1 inhibitor Ac-YVAD-CHO (Fig. 1A). We then tested the possibility that IL-33 may also be a substrate for other caspases. We found that incubation of in vitro translated full-length IL-33 protein with caspase-3, the prototypic apoptotic caspase, led to the production of a 20–22 kDa fragment very similar in size to the fragment observed after caspase-1 cleavage (Fig. 1B). Like cleavage by caspase-1, cleavage of IL-33 by caspase-3 was specific, as it was not observed in the presence of the caspase-3 inhibitor Ac-DEVD-CHO. We concluded that full-length IL-331–270 protein can be cleaved by both caspase-1 and caspase-3 in vitro and that processing results in both cases in the generation of a 20–22 kDa cleavage product.

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 in A. 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 in A and analyzed by fluorography (fluorescent IL-33) or Western blot (unlabeled IL-33) with IL-33 Nter, Nessy-1, or anti-myc antibodies.

Schmitz et al. (4) suggested caspase-1 cleaves IL-331–270 after residue Ser111, resulting in the production of mature IL-33112–270, corresponding to the C-terminal IL-1-like cytokine domain. Unexpectedly, we discovered that the 20–22 kDa caspase-1 cleavage product does not correspond to the C-terminal IL-1-like domain, as initially proposed. Western blot analysis with antibodies specific for the N- and C-terminal domains of IL-33 [see supporting information (SI) Fig. S1] clearly revealed that antibodies directed against the first 15 amino-terminal residues of IL-33 (IL-33-Nter) recognize the 20–22 kDa caspase-1 cleavage product, whereas the Nessy-1 mAb, which is directed to the C-terminal IL-1-like domain (IL-33179–270), recognizes a smaller fragment of 12–13 kDa, migrating close to the gel front and found at very low levels compared with the full-length IL-33 protein (Fig. 1C). We used an IL-33 protein tagged with myc-epitope at its carboxy-terminus in these experiments, and Western blot analysis with an anti-myc mAb detected the small 12–13 kDa fragment but not the 20–22 kDa form (Fig. 1C), allowing us to independently confirm that the 20–22 kDa caspase-1 cleavage product corresponds to the N-terminal, rather than C-terminal, part of IL-33.

Caspase-1 Cleaves IL-33 After Residue Asp178 Within the IL-1-Like Cytokine Domain.

The size of the N-terminal fragment generated after caspase-1 processing indicated to us that cleavage must occur within the IL-1-like domain. Inspection of this region revealed the existence of a potential cleavage site (DGVD178G), similar to the consensus site of cleavage by caspase-3, and located between the fourth and fifth predicted β-strands of the IL-1-like domain (Fig. 2A). The possibility that caspase-1 may cleave IL-33 at this position was further supported by the observation that an in vitro translated fragment corresponding to IL-33 residues 1–178 co-migrated on SDS/PAGE with the 20–22 kDa caspase-1 cleavage product (Fig. 2B). To provide definitive proof for caspase-1 cleavage of IL-33 after residue Asp178, a single mutation was introduced that replaced Asp178 by an alanine. As shown in Fig. 2C, this mutation totally prevented the processing of full-length IL-33 by caspase-1. We concluded that caspase-1 cleaves IL-331–270 at the DGVD178G site. Mutation of Asp178 to alanine also abrogated cleavage of IL-33 by caspase-3 (Fig. 2D), indicating that caspase-1 and caspase-3 cleave IL-331–270 at the same site within the IL-1-like domain. We then asked whether cleavage of IL-33 at this site occurs in cells undergoing apoptosis. WT IL-33 and the IL-33D178A mutant were expressed in U2OS epithelial cells (Fig. S2), and apoptosis was induced by treatment with the DNA-damaging agent doxorubicin. Western blot analysis revealed that WT IL-33, but not the IL-33D178A mutant, is cleaved during doxorubicin-induced apoptosis and that cleavage can be prevented by pretreatment with the pan-caspase inhibitor Z-VAD-fmk (Fig. 2E). We next looked at endogenous native IL-33. As endothelial cells constitute a major cellular source of IL-33 in human tissues (20, 21), we selected human primary endothelial cells as a cellular system for these experiments. Western blot analysis revealed that endogenous IL-33 migrates as a 30–31 kDa band, the identity of which was validated after knockdown of IL-33 expression with specific siRNAs (Fig. 2F). Treatment of the endothelial cells with the apoptosis-inducing agent staurosporine resulted in a complete maturation of endogenous IL-33 that was prevented by pretreatment with the pan-caspase inhibitor Z-VAD-fmk (Fig. 2G). We concluded that native IL-33 is processed by endogenous caspases during apoptosis in primary human endothelial cells.

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 and D) 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 in B. 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).

Full-Length IL-331–270 Binds and Activates the IL-33 Receptor ST2.

As our data indicated that, unlike IL-1β and IL-18, cleavage of IL-33 by caspase-1 does not lead to the production of the mature IL-1-like domain, we then asked whether, similarly to the IL-1α precursor (1, 31), full-length IL-331–270 may possess biological activity. We first tested the capacity of full-length IL-331–270 to bind to the ST2 receptor in pull-down experiments. We found that a human ST2-Fc fusion protein precipitates in vitro synthesized full-length IL-331–270, similarly to the isolated IL-1-like domain IL-33112–270 (Fig. 3A). In contrast, the amino-terminal part of IL-33, IL-331–111, did not bind to the ST2-Fc chimera protein. These results indicated that full-length IL-331–270 specifically binds to the ST2 receptor. Binding of full-length IL-33 to ST2 was also observed using endogenous native IL-33 obtained from endothelial cells extracts (Fig. 3B), indicating the natively folded forms behave similarly to the in vitro generated forms regarding ST2 binding.

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 and D) 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 in Materials 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.

We next investigated the capacity of full-length IL-331–270 to signal through ST2 by using a previously described NF-κB-dependent reporter assay (4, 14). HEK293T cells, transiently transfected with an expression vector for human ST2 together with an NFκB-GFP reporter gene construct, were stimulated with full-length IL-331–270, IL-331–111, or IL-33112–270. As previously reported (4, 14), a small population of cells expressed GFP in the absence of IL-33 stimulation. However, stimulation of cells with full-length IL-331–270 or the IL-1-like domain IL-33112–270, but not IL-331–111, led to a significant increase in the number of GFP+ cells (Fig. 3C). Quantification of the results by FACS analysis revealed that full-length IL-331–270 and the IL-1-like domain IL-33112–270 possess a similar capacity to activate the NFκB-GFP reporter gene construct (Fig. 3D). We concluded that, similarly to IL-33112–270, full-length IL-331–270 is able to bind and activate the ST2 receptor.

The 2 Caspase-1 Cleavage Products, IL-331–178 and IL-33179–270, Do Not Activate ST2.

As we found caspase-1 processing of IL-33 occurs within the IL-1-like domain, we predicted the 2 caspase-1 cleavage products, IL-331–178 and IL-33179–270, would not exhibit biological activity. To confirm this prediction, we tested the capacity of IL-331–178 and IL-33179–270 to activate ST2 using the NFκB-dependent GFP reporter assay. Immunofluorescence (Fig. 4A) and FACS (Fig. 4B) analyses revealed that, whereas treatment of the cells with full-length IL-331–270 resulted in significant activation of ST2-dependent signaling, treatment with the 2 caspase-1 cleavage products did not activate the ST2-dependent NFκB-GFP reporter gene. These results were confirmed using another bioassay, IL-33-dependent secretion of IL-6 by the mast cell line MC/9 (32). Full-length IL-33 significantly induced IL-6 secretion by MC/9 cells whereas the 2 caspase-1 cleavage products had no effect (Fig. 4C). We concluded that, unlike full-length IL-331–270. the 2 caspase-1 cleavage products, IL-331–178 and IL-33179–270, do not possess biological activity, indicating that caspase-1 processing inactivates IL-33.

Fig. 4.

Fig. 4.

The 2 caspase-1 cleavage products, IL-331–178 and IL-33179–270, are not able to activate ST2. (A and B) 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 in Materials 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.

IL-33 Is Released Extracellularly After Endothelial Cell Damage or Injury.

IL-33 and the alarmin HMGB1 are both chromatin-associated cytokines, and we then studied the possibility that full-length IL-33, similarly to HMGB1 (26), may be released after cell damage or necrosis. We first tested the effect of mechanical injury on primary human endothelial cells that express high levels of endogenous IL-33 both in vivo (20, 21) and in culture (Fig. 5A). Mechanical wounding of endothelial cells by cell scraping, a process that mimics the transient sublethal membrane disruptions that are observed in cells subjected to mechanical forces in vivo, has previously been shown to result in the release of growth factors such as basic FGF (33). Interestingly, we found that full-length IL-33 was released from endothelial cells mechanically wounded by cell scraping (Fig. 5B). HMGB1 was similarly released under these conditions. Scratching of endothelial monolayers by tracing lines with a surgical scalpel, a needle, or a pipette tip has been widely used as an in vitro model of wound healing. Using this model, we confirmed that IL-33, like HMGB1, is released into the supernatant after mechanical injury of endothelial cells. In contrast, IL-33 and HMGB1 were not released extracellularly in the absence of endothelial cell damage (Fig. 5B). We then tested the effect of necrosis induced by several cycles of freezing and thawing (26), and found IL-33 was released by necrotic cells and its levels in the supernatant increased with the number of freezing-thawing cycles (Fig. 5C). Finally, full-length IL-33 and HMGB1 were also detected in the medium of endothelial cells treated with non-ionic detergents Nonidet P-40 and Triton X-100, which provoke damage to the cell membrane (Fig. 5D). Together, these data demonstrate that IL-33, like HMGB1, can be released in the extracellular space after endothelial cell damage or mechanical injury.

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.

Discussion

In the present study, we demonstrate that IL-33 does not require maturation for binding and activation of the IL-33 receptor ST2, and that, contrary to the current belief, processing by caspase-1 results in IL-33 inactivation, rather than activation. Indeed, we unexpectedly discovered that cleavage of IL-33 by caspase-1 does not occur at the site initially proposed (Ser111), but rather at a site located in the middle of the IL-1-like domain (DGVD178G). To the best of our knowledge, IL-33 is the first member of the IL-1 family shown to be inactivated after maturation by caspase-1. IL-33 thus differs from IL-1β and IL-18, which require maturation by caspase-1 for liberation of their mature biologically active forms (1, 2); from IL-1 family member IL-1F7, which requires caspase-1 processing for translocation to the nucleus (34); and from IL-1α, which is not a substrate for caspase-1 but is cleaved by calpain to release a C-terminal 17 kDa form with biological activity (1). IL-33 is therefore very unique in the IL-1 family, in terms of processing and biologically active domains. As our data convincingly demonstrate caspase-1 cleaves IL-33 at position D178 but not at position S111, and no protease is known to cleave after S111, there is no evidence left for the existence of IL-33112–270 either in vitro or in vivo.

The caspase-1 processing site within IL-33 is very similar to a consensus site for caspase-3, the pro-apoptotic caspase, and we found that IL-33 is cleaved at this site by caspase-1 and caspase-3 in vitro and by endogenous caspases in cells undergoing apoptosis. IL-33 is a potent stimulator of pro-inflammatory cytokine production by mast cells and other cells of the innate immune system (10, 11, 15, 18, 19). Processing of IL-33 by caspases may therefore provide a mechanism to inactivate IL-33 pro-inflammatory cytokine activities during apoptosis, a process that does not trigger inflammation in vivo.

Similarly to IL-1α (1, 27–29), IL-33 may be released by damaged cells in vivo and function as an endogenous danger signal (35), or alarmin (30), to alert cells of the innate immune system of tissue injury. This possibility is supported by our data indicating that IL-33, which is constitutively expressed to high levels by endothelial cells in most normal human tissues (20, 21), can be released in the extracellular space after endothelial cell damage or mechanical injury. Interestingly, IL-1α and the prototypical alarmin HMGB1 have also been shown to be released from damaged endothelial cells (28), suggesting the endothelium may constitute a major source of danger signals in vivo. In addition to endothelium, we also detected abundant expression of IL-33 in the nucleus of epithelial cells of tissues in contact with the environment, including the skin and gastrointestinal tract, where pathogens, allergens, and other environmental agents are frequently encountered (20). The constitutive expression of IL-33 in epithelial barriers of the human body thus supports the possibility that IL-33 may also play important roles as an epithelial alarmin. IL-33 is likely to be a very good alarm signal because it has the capacity to activate many actors of the innate immune system, including mast cells, basophils, eosinophils, NK cells, and invariant NK T cells (4, 10–13, 15–19). Among these, mast cells, which are strategically positioned close to vessel walls and epithelial surfaces exposed to the environment (36), the major sites of IL-33 expression in vivo (20) may play a major role in the response to the IL-33 danger signal. IL-33 is also able to activate Th2 cells (4, 14, 17, 18) and it may therefore play a role not only in the innate response, but also in the adaptive immune response, after tissue damage. Another property of IL-33 that qualifies it as a bona fide danger signal is the fact IL-33 signals through the TLR/IL-1R/MyD88 signaling pathway (4, 13, 17), like IL-1α (27) and the exogenous danger signals from microorganisms, the so-called pathogen-associated molecular patterns. IL-33 may therefore represents a novel member of the damage-associated molecular pattern family, which regroups both endogenous (ie, alarmins) and exogenous (ie, pathogen-associated molecular patterns) danger signals (30).

In summary, the results presented here are important because they demonstrate that IL-33 does not require maturation for biological activity and that, contrary to the current view, processing by caspase-1 results in IL-33 inactivation, rather than activation. In addition, the data reveal that full-length IL-33 can be released after endothelial cell damage or injury, suggesting IL-33 may function as a novel endogenous danger signal. Future studies will aim at defining the precise functions of IL-33 in human health and disease, including its roles in the tissue response to trauma or infection.

Materials and Methods

Plasmid Constructions and Protein Production.

IL-33 deletion mutants were amplified by PCR using the human IL-33/NF-HEV cDNA (NM_033439) (3, 5) as a template. The PCR fragments thereby obtained were cloned into plasmid pcDNA3.1A/myc-his (Invitrogen). The IL-33D178A mutant was generated by PCR and cloned into the same expression vector. IL-331–270 and IL-331–178 deletion mutant were also cloned into plasmid pcDNA3 (Invitrogen) for use in experiments presented in Figs. 2, 3B, and 5B. Human IL-1β (NM_000576.2) was amplified by PCR and cloned into vector pcDNA3.1A/myc-his. All primer sequences are available upon request. Pro-IL1β, full-length IL-331–270, IL-33D178A, and IL-33 deletion mutants were synthesized in vitro in rabbit reticulocyte lysate using the TNT-T7 kit with (fluorescent protein) or without (unlabeled protein) the FluoroTect GreenLys labeling system according to the manufacturer's instructions (Promega).

In Vitro Caspase Cleavage Assays. In vitro translated fluorescent or unlabeled proteins (5 μL lysate) were incubated with various amounts of recombinant caspase-1 (Sigma) or caspase-3 (Calbiochem) in 14 μL assay buffer (Calbiochem) for 2 h at 37 °C. The resulting cleavage products were analyzed by SDS/PAGE and fluorography (Typhoon 9400 fluoroimager; GE Healthcare) or Western blot. In some experiments, caspases 1 and 3 were preincubated for 20 min at 37 °C with 100 μM of their respective inhibitors, Ac-YVAD-CHO and Ac-DEVD-CHO (Calbiochem).

Western Blot and Pull-Down Assays.

Proteins were fractionated by SDS/PAGE, electroblotted, and detected with mAbs to myc-epitope tag (9E10, 1:1,000; Sigma), IL-33 (Nessy-1, 1:1,000; Alexis Biochemicals; 305B, 1:1,000; Alexis Biochemicals), or PARP (1:2,000; BD PharMingen), or rabbit antiserum to IL-33-Nter (IL-331–15, 1:400) (3, 5), IL-33-Cter (AT-110, no. 210–447, 1:1,000; Alexis Biochemicals), or HMGB1 (Ab18256, 1:200; Abcam), followed by HRP-conjugated goat anti-mouse or anti-rabbit Ig (1:10,000; Promega), and finally an enhanced chemiluminescence kit (GE Healthcare). Pull-down assays with human ST2-Fc chimera protein (1 μg; R&D Systems) were performed as previously described (4), using in vitro translated IL-33 proteins (25 μL lysate) or endothelial cell freeze-thaw extracts (3.5 × 106 cells) containing endogenous native IL-33.

Reporter Gene Assays and ELISA.

HEK293T cells (1.5 × 105 cells/well in 12-well plates) were transfected with 2 μg pNF-κB-hrGFP (Stratagene) reporter plasmid and 1 μg of pEF-BOS-hST2 (provided by S. Tominaga, Tochigi, Japan), using a phosphate calcium precipitation method. One day after transfection, cells were stimulated for 16 h with in vitro translated full-length IL-33 or deletion mutants (12.5 μL lysate/well). Cells were then analyzed for GFP expression by fluorescence microscopy (Eclipse TE300 microscope; Nikon) and flow cytometry (FACScan, Cellquest Software; Becton Dickinson). DuoSet IL-6 ELISA assays (R&D Systems) were performed as described (32) using MC/9 mast cells (ATCC; 4 × 105 cells/well in 96-well plates) stimulated for 40 h with in vitro translated full-length IL-33 or deletion mutants (10 μL lysate/well).

Mammalian Cell Culture and Induction of Apoptosis or Cell Damage.

Human HEK293T and U2OS epithelial cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (all from Invitrogen). Human umbilical vein endothelial cells (Praxcell) were cultured in endothelial cell growth medium (Promocell), supplemented with 20% fetal calf serum (Invitrogen) and heparin (Sigma), and used at confluence in all experiments. Knockdown of IL-33 expression with ON-TARGET plus SMARTpool siRNA duplexes (Dharmacon) was performed as described (22). For induction of apoptosis, cells were incubated with 1 μM staurosporine (Sigma) for 3 h (endothelial cells) or 2 μM doxorubicin (Sigma) for 24 h (U2OS cells), with or without pretreatment with 50 μM pan-caspase inhibitor Z-VAD-fmk (Calbiochem) for 16 h (endothelial cells) or 24 h (U2OS cells). In other experiments, cell damage was induced by repeated cycles of freezing and thawing (26), mechanical scraping from the substratum (33), or mechanical wounding of monolayers by tracing lines with a surgical scalpel. Supernatants were prepared from treated and untreated cells in Opti-MEM serum-free media (Invitrogen), by spinning the cells or cell lysates at 16000× g for 5 min. In some cases, supernatants were concentrated by TCA or filtration using Vivaspin columns (Sartorius).

Supplementary Material

Supporting Information

Acknowledgments.

We thank Drs. Tominaga and Yanagisawa (Tochigi, Japan) for the gift of hST2 expression vector. This work was supported by grants from Ligue Nationale contre le Cancer (Equipe labellisée Ligue 2009), ANR-Program Blanc “Cuboïdale,” and MAIN European Network of Excellence (FP6–502935).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

References

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information