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Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity *

Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 11, pp. 8205–8213, March 9, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity Received for publication, August 19, 2011, and in revised form, January 12, 2012 Published, JBC Papers in Press, January 23, 2012, DOI 10.1074/jbc.M111.295055 ‡1 §1 ‡1 ‡ ‡ ‡ ‡ Suyoung Bae , Taebong Kang , Jaewoo Hong , Siyoung Lee , Jida Choi , Hyunjhung Jhun , Areum Kwak , ‡ ‡ ‡ ‡2 Kwangwon Hong , Eunsom Kim , Seunghyun Jo , and Soohyun Kim From the Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701 and the Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chung-Ju 380-701, Korea Background: The maturation process of IL-33 (IL-1F11) remains unclear. Results: IL-33 ligand affinity column isolates neutrophil proteinase 3. Conclusion: PR3 is an IL-33-processing enzyme. Significance: PR3 has a dual function in IL-33 biological activity. IL-1 family ligand does not possess a typical hydrophobic type immune responses like immunity against nematodes and signal peptide and needs a processing enzyme for maturation. allergic diseases (2–6), mast cells (4, 7), basophils (8), and eosin- The maturation process of IL-33 (IL-1F11), a new member of ophils (5, 9, 10). IL-33 also induces non-Th2 inflammatory the IL-1 family ligand, remains unclear. Precursor IL-33 cytokines such as TNF, IL-1, or IL-6 (11–13). It has also been ligand affinity column isolates neutrophil proteinase 3 (PR3) suggested that the properties of IL-33 are cytokines or a nuclear from human urinary proteins. PR3 is a known IL-1 family transcription factor like IL-1 and high mobility group protein ligand-processing enzyme for IL-1 (IL-1F2) and IL-18 (IL- B1 (4, 5, 14–23). 1F4), including other inflammatory cytokines. We investi- ST2 (also known as IL-1RL1, DER4, Fit-1, or T1), which was gated PR3 in the maturation process of precursor IL-33 originally discovered as an orphan receptor (24–28), is a ligand because we isolated urinary PR3 by using the precursor IL-33 binding chain of IL-33 (6). IL-33 receptor complex for signaling ligand affinity column. PR3 converted inactive human and is composed of the ligand chain ST2 and signal transducing mouse precursor IL-33 proteins to biological active forms; chain IL-1 receptor accessory protein (IL-1RAcP) (2, 6, 29). however, the increase of PR3 incubation time abrogated This receptor complex activates downstream signaling mole- IL-33 activities. Unlike caspase-1-cleaved precursor IL-18, cules such as NF-B and AP-1 through IL-1 receptor-associ- PR3 cut precursor IL-33 and IL-18 at various sites and yielded ated kinase, TNF receptor-associated factor 6, and/or MAPKs multibands. The increased incubation period of PR3 abated (6). These cells produce inflammatory cytokines and chemo- mature IL-33 in a time-dependent manner. The result is con- kines, including IL-4, IL-5, IL-6, IL-13, and IL-8, by stimulation sistent with the decreased bioactivity of IL-33 along with the of IL-33 (5, 7, 9, 30). Recently, it has been reported that circu- increased PR3 incubation time. Six different human and lating CD34 hematopoietic progenitors expressed ST2 and mouse recombinant IL-33 proteins were expressed by the responded to IL-33 by releasing high levels of Th2-associated predicted consensus amino acid sequence of PR3 cleavage cytokines (31). Such results suggest potential roles of IL-33 in sites and tested for bioactivities. The human IL-33/p1 was Th2-associated immune responses, and IL-33 seems to be highly active, but human IL-33/p2 and p3 proteins were inac- closely related to allergic inflammatory diseases, including tive. Our results suggest the dual functions (activation/termi- asthma and atopic dermatitis. nation) of PR3 in IL-33 biological activity. and IL-18, is produced as a precur- IL-33, similarly to IL-1 sor IL-33 molecule. This precursor IL-33 does not have a signal peptide to be secreted; it is released extracellularly as a mature IL-33 is a new member of the IL-1 family ligand and was protein after cleavage (6). It is known that inflammatory originally discovered as a nuclear factor from high endothelial and IL-18 (32, caspases are necessary for the cleavage of IL-1 venules (1). IL-33 is considered to be critical in inducing Th2- 33). Although it has been suggested that precursor IL-33 is pro- cessed by caspase-1 (6), the exact role of caspases in IL-33 biol- * This work was supported by the National Research Foundation funded by ogy still remains unclear (34, 35). Korean Government Grants WCU R33-2008-000-10022-0 and KRF-2008- In this study, we identified and characterized a processing 313-C00644 and Korea Healthcare Technology R&D Project, Ministry of enzyme of precursor IL-33. PR3 converts inactive precursor Health & Welfare, Republic of Korea Grant A100460. These authors contributed equally to this work. IL-33 to active form, whereas a longer incubation period of PR3 To whom correspondence should be addressed: Laboratory of Cytokine abolishes IL-33 activity. This result suggests the contrasting Immunology, Dept. of Biomedical Science and Technology, Konkuk Uni- functions (activation/termination) of PR3 in precursor IL-33 versity, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701 Korea. Tel.: 82-2- 457-0868; Fax: 82-2-2030-7788; E-mail: [email protected]. bioactivity. MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8205 This is an Open Access article under the CC BY license. Interleukin-33 Processing Enzyme MATERIALS AND METHODS ance liquid chromatography (GE Healthcare) with C4 column (Grace Vydac, Hesperia, CA). The two-step purified recombi- RT-PCR and Molecular Cloning for Escherichia coli Expres- nant proteins were tested for endotoxin level (below 0.25 EU sion Vectors—Total RNA was isolated with TRI Reagent per g of recombinant protein) by using LAL method (Cape (Sigma) from human PC-3 cells and mouse lung tissue. A pair of Cod, East Falmouth, MA) according to the manufacturer’s human IL-33 sense primers 5-ACAGAATACTGAAAAATG- instructions and then used for experiments. AAGCC-3 and reverse primer 5-CTTCTCCAGTGGTAGC- Caspase-1 and PR3 Cleavage Test—Caspase-1 (10 units from ATTTG-3, mouse IL-33 sense primers 5-ATGAGACCTAG- Millipore, Temecula, CA) and the recombinant proteins of pre- AATGAAGTATTC-3 and reverse primer 5-GCACAGGCG- cursor human IL-33, mouse IL-33, and human IL-18 (500 ng) TTTTACTGCATT-3, and -actin sense primers 5-ACCAA- were incubated in 20 l of a reaction buffer (25 mM K HEPES, CTGGGACGACATGGA-3 and reverse primer 5-GTGATG- 1mM DTT, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, pH 7.5) at ACCTGGCCGTCAGG-3 was used for the RT-PCR. Moloney 37 °C for 30 min. After the reaction, the sample was subjected to murine leukemia virus-RT (Beams Bio, Korea) was used for 10% SDS-PAGE for silver staining. converting 2 g of total RNA to first strand cDNA, and then For PR3 cleavage test with different IL-1 family ligands, com- PCR was performed at 94 °C for 45 s, 70 °C for 2 min, and 59 °C mercial PR3 (100 ng from Athens Research & Technology) and for 1 min for 30 cycles. the recombinant proteins of precursor human IL-33, mouse For E. coli expression vector, the PCR product of human and IL-33, and human IL-18 (500 ng) were incubated in 20 lofa mouse precursor IL-33 cDNA was ligated into T&A cloning reaction buffer (50 mM Tris, pH 8) at room temperature. The vector (RBC, Taiwan). We designed the sense primer with an PR3-cleaved samples were subjected to 10% SDS-PAGE for sil- EcoRI (GAATTC) site and the reverse primer with a KpnI ver staining. (GGTACC) site to transfer the amplified PCR product into Western Blots—For the detection of PR3-cleaved human pProEX/HTa (Invitrogen). rIL -33, the samples were loaded on 10% SDS-PAGE. Anti-hu- The insert of human precursor IL-33, IL-33/p2, and IL-33/p3 man IL-33 polyclonal antibody was developed by immunizing was amplified with the same sense primer (5-GAACGAATT- three BALB/c mice (6 weeks old) with mature human IL-33 CATGAAGCCTAAAATGAAG-3). The insert of human pre- 112 270 (Ser –Thr ) protein (data not shown). Human IL-33 poly- cursor IL-33 and IL-33/p1 was amplified with the same reverse clonal antibody was used for detecting a mature size of IL-33, primer (5-GAACGGTACCCTAAGTTTCAGAGAGCTT- which was cleaved by PR3. In addition, an anti-human PR3 3). The sense primer of human IL-33/p1 (5-TAAAGAATTC- monoclonal antibody (36) was obtained from YbdYbiotech ACCTATTACAGAGTATCTT-3) and the reverse primer of (Seoul, Korea) and used for detecting urinary PR3. Peroxidase- IL-33/p2 (5-TATTGGTACCTTAGACAAAGAAGGCCTG- conjugated goat anti-mouse IgG secondary antibody (Jackson 3) and IL-33/p3 (5-TATTGGTACCTTATATAAACACTC- ImmunoResearch) was used to develop the blots by using Supex CAGG-3) were used to amplify cDNA inserts. (Neuronex, Korea) and LAS-4000 imaging device (Fujifilm, For mouse precursor IL-33 and IL-33/p1, the same reverse Japan). primer (5-TATTGGTACCTTAGATTTTCGAGAGCTT-3) For detecting the phosphorylation of signaling molecules and the sense primer of mouse precursor IL-33 (5-TAAAG- (IRAK1, NF-B, p38 MAPK, p44/42 MAPK, and JNK), AATTCATGAGACCTAGAATGAAG-3) and IL-33/p1 (5- HMC-1 and Raw 264.7 cells were stimulated with precursor TAAAGAATTCTCACTTTTAACACAGTCT-3) were used and mature IL-33 at various time points. Cells were lysed to amplify cDNA inserts. The PCR product of a single band with kinase lysis buffer (Cell Signaling Technology, Beverly, from each construct was digested with EcoRI and KpnI and MA) and then subjected to 10% SDS-PAGE. The samples then transferred into pProEX/HTa (Invitrogen) for recombi- were transferred to nitrocellulose membranes. The mem- nant protein expression in E. coli. branes were blocked in 3% BSA/TBST (Santa Cruz Biotech- Isolation of Urinary IL-33 Interacting Molecule from Concen- nology, Santa Cruz, CA). The membranes were probed first trated Human Urine—Human precursor IL-33 (3 mg) was with rabbit anti-phospho-IRAK1, mouse anti-phospho-NF- immobilized by coupling to Affi-Gel-15 beads according to the B, rabbit anti-phospho-p38 MAPK, mouse anti-phospho- manufacturer’s instructions (Bio-Rad). Batches of 300 ml of p44/42 MAPK, or rabbit anti-phospho-JNK (Cell Signaling crude urinary proteins concentrated 500-fold were passed over Technology). The membranes were reprobed with rabbit the precursor IL-33-bound beads at 4 °C. The column was anti-NF-B, p38 MAPK (Santa Cruz Biotechnology), p44/42 washed with 500 ml of phosphate buffer containing 0.5 MAPK, JNK, or IRAK1 (Cell Signaling Technology) for nor- sodium chloride, pH 8. Bound proteins were eluted in a 1-ml malization of each protein and then normalized finally with volume of solution containing 25 mM citric acid, pH 2.2, and goat anti-actin (Santa Cruz Biotechnology). then the eluted fraction was immediately neutralized with 2 M Cell Culture and Bioassay—Mouse macrophage Raw 264.7 glycine. cell line was obtained from American Type Culture Collection Generation of Recombinant Protein—Six recombinant (hu- (ATCC) and maintained according to the instructions. HMC-1 man and mouse precursor IL-33, human and mouse IL-33/p1, cells were cultured in Iscove’s modified Dulbecco’s medium and human IL-33/p2, IL-33/p3) proteins were expressed in enriched with 10% FBS. For a blockade of ST2, a fresh medium E. coli Rosetta cells (Novagen, Madison, WI) and purified by a TALON affinity column (Invitrogen). The TALON affinity-pu- rified recombinant proteins were subjected to high perform- The abbreviation used is: rIL, recombinant IL. 8206 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 11 •MARCH 9, 2012 Interleukin-33 Processing Enzyme FIGURE 1. Isolation of an IL-33 interacting molecule by using ligand affinity column. A, precursor IL-33 affinity chromatography column isolated urinary IL-33-interacting proteins and was visualized by silver-stained 10% SDS-PAGE. Four fractions were eluted from the IL-33 ligand affinity column. Molecular mass is indicated on the left. The arrows indicate the IL-33-binding proteins, 56-, 28-, and minor 36-kDa bands. B, mouse anti-human PR3 antibody was used for verifying urinary PR3 in the fractions from the IL-33 ligand affinity column. The molecular sizes of56 (dimer form) and 28 kDa (monomer form), including the minor 36-kDa (precursor PR3) bands, were detected in the fractions. The data represent one of three independent experiments. (0.2 ml) containing various concentrations of rIL-33 and 0.5 g of anti-ST2 (R&D Systems) was used. Human HMC-1 and mouse Raw 264.7 cells were treated with different stimuli, and the cell culture supernatant was harvested the next day for cyto- kine assay. Measurement of Cytokine Level—Human IL-8, mouse TNF, mouse MIP-2, and mouse IL-6 ELISA kits were obtained from R&D Systems. Cytokine levels were measured in culture super- natants by sandwich ELISA according to manufacturer’s FIGURE 2. PR3 enhancing precursor IL-33-induced cytokine production. instructions. A, PR3-preincubated precursor IL-33 induced IL-8 in a dose-dependent man- Statistical Analysis—The data are expressed as means  S.E. ner. PR3 (25 ng/ml) was preincubated for 5 min with various concentrations of Statistical significance of differences were analyzed by precursor IL-33 as indicated on the bottom and then used for stimulating HMC-1 cells. B, precursor IL-33 (100 ng/ml) activity was gradually decreased unpaired, two-tailed Student’s t test. Values of p  0.05 were along with increasing PR3 incubation times. The data represent one of three considered statistically significant. independent experiments. cont, control. RESULTS and U937), and the molecular size of precursor PR3 is similar Isolation of an IL-33 Interacting Molecule from Urinary to the 36-kDa band (data not shown). Proteins—In an attempt to isolate a soluble IL-33 interacting PR3 Converted Precursor IL-33 into Mature IL-33—The bio- molecule, concentrated urine was applied to a column com- logical activity of human and mouse precursor IL-33 was exam- posed of precursor IL-33 covalently immobilized to agarose ined in the presence of PR3. The pretreatment of PR3 activated beads. After extensive washing with PBS, interacting pro- precursor IL-33 and induced IL-8 production by a dose-depen- teins were eluted at low pH citric acid solution. Aliquots of dent manner, but precursor IL-33 alone did not induce IL-8 the various fractions were resolved by 10% SDS-PAGE, and production (Fig. 2A). The time course of PR3 pretreatment the protein bands were visualized by silver staining. The revealed that PR3 induced the highest activity of IL-33 at 5 min, broad bands corresponding to specific IL-33 interacting and its activity was decreased along with increasing incubation proteins, 56- and 28-kDa bands including the minor times (Fig. 2B). PR3 pretreatment for 30 min completely abol- 36-kDa band, were detected mainly in fractions 2 and 3 (Fig. ished IL-33 activity. 1A). The 56- and 28-kDa protein bands in elution fraction 3 Examination of IL-33 Cleavage by PR3—We tested the cleav- were excised and analyzed by mass spectrometer analysis. age of human and mouse precursor IL-33 proteins with The result of mass spectrometer revealed that the bands caspase-1 and PR3. Caspase-1 specifically cleaved precursor were neutrophil proteinase 3 (PR3). We verified urinary PR3 IL-18 resulting in the production of 18-kDa mature IL-18, in the eluted fractions with a monoclonal antibody specific which was active in the A549R cell line (data not shown) (37), to human PR3. The anti-human PR3 monoclonal antibody but precursor IL-33 proteins were not cleaved by caspase-1 recognized the 56-, 36-, and 28-kDa urinary PR3 and com- (Fig. 3A). Next we examined the cleavage of precursor IL-33 mercial PR3 (Fig. 1B). The results suggest that 28 and 56 kDa proteins with PR3. As shown in Fig. 3B, PR3 cleaved the pre- are the monomer and dimer forms of mature PR3, respec- cursor IL-33 and produced multibands unlike caspase-1- tively. The additional minor 36-kDa band could be the pre- cleaved IL-18 (Fig. 3A). PR3 also cleaved precursor IL-18 as cursor PR3 because the anti-PR3 monoclonal antibody rec- multiband products in silver staining (Fig. 3B, 2nd lane from ognized the precursor PR3 in human monocyte cells (THP-1 right). MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8207 Interleukin-33 Processing Enzyme FIGURE 3. Silver staining of caspase-1 and PR3-cleaved precursor IL-33. A, process of precursor human IL-33 (250 ng/lane), mouse IL-33 (100 ng/lane), and IL-18 (500 ng/lane) was examined with caspase-1 (Casp-1) (10 units/lane). The preincubation of caspase-1 specifically cleaved precursor IL-18 and produced a single band of mature IL-18, molecular mass of 18 kDa, but both human and mouse precursor IL-33 were not affected. B, same amount of recombinant IL-1 family ligands was used for testing if these ligands were processed by PR3 (100 ng/lane). Unlike caspase-1, PR3 cleaved precursor IL-18 and produced multibands in the 2nd lane from right. The pattern of PR3-cleaved precursor IL-33 was very similar in both human and mouse. The data represent one of three independent experiments. predicted by the consensus sequence of the PR3 cleavage site. The predicted PR3 cleavage sites are indicated in red as p1, p2, and p3 (Fig. 5A). The previously reported caspase cleavage sites are shown in blue (Fig. 5A). We expressed six rIL-33 proteins as shown at the top of Fig. 5B. The purified rIL-33 proteins were subjected to 10% SDS-PAGE, and the result showed a single band of correct molecular size of each recombinant protein (Fig. 5B). The upper bands of precursor IL-33 and IL-33/p2–3 are dimer forms that were verified by Western blot (data not shown). Additional experiments tested the biological activity of 112 270 109 mature human IL-33 (Ser –Thr ) and mouse IL-33 (Ser – Ile ) in the presence or absence of the N-terminal His tag. The N-terminal His tag did not affect the bioactivities of mature human and mouse rIL-33 (data not shown). Biological Activity of rIL-33/p1 Proteins—Four human IL-33 proteins (Fig. 5B) were used to test the biological activities with 117 270 FIGURE 4. Time-dependent reduction of a mature size of IL-33. Time human HMC-1 cells. IL-33/p1 (Ser –Thr ) was highly course study of human precursor (Pro) IL-33 (100 ng/lane) cleavage was per- active and induced IL-8 production, but precursor IL-33 formed in the presence of PR3 (20 ng/lane). The precursor IL-33 was promptly 1 270 1 220 (Met –Thr ), huIL-33/p2 (Met –Val ), and huIL-33/p3 reduced after 5 min of PR3 incubation. Western blot revealed that a mature 1 240 (Mat) size of IL-33 was decreased by a time-dependent manner. The data (Met –Ile ) were not active (Fig. 6A). We repeated cytokine represent one of three independent experiments. assay with mouse Raw 264.7 cells, and mouse TNF production was consistent with that of IL-8 (Fig. 6B). We did not produce Time-dependent Reduction of a Mature Size IL-33 by PR3— the mouse form of IL-33/p2 and IL-p3 proteins because both IL-33 protein was incubated at different time points to examine huIL-33/p2 and huIL-33/p3 were not active (Fig. 6). whether the multiband products of the PR3 cleaved is a mature We continued to produce the large scale of human rIL-33/p1 IL-33. We used the anti-IL-33 antibody to detect mature IL-33. 117 270 114 266 (Ser –Thr ) and mouse rIL-33/p1 (Ser –Ile ) because The result of the Western blot exhibited that PR3-cleaved human rIL-33/p1 was only active in the cytokine assay (Fig. 6). mature IL-33 was highest at 5 min and decreased gradually 117 270 Human IL-33/p1 (Ser –Thr ) has six amino acids less than along with incubation time (Fig. 4). This result is consistent 111 270 the originally proposed mature human IL-33 (Ser –Thr ) with bioactivity of the PR3-incubated precursor IL-33 in Fig. by caspase-1 cleavage (6). The biological activity of human 112 270 2B. Mature rIL-33 (Ser –Thr ) was completely degraded by 117 270 114 266 rIL-33 (Ser –Thr ) and mouse rIL-33/p1 (Ser –Ile ) PR3 after 30 min of incubation (Fig. 4, far right lane). was compared with commercial human mature IL-33 (Ser Design and Expression of PR3-cleaved rIL-33 Proteins—We 270 109 266 Thr ) and mouse mature IL-33 (Ser –Ile ) from R&D Sys- aligned the amino acid sequence of human and mouse IL-33 to tems. Our human and mouse IL-33/p1 proteins induced TNF predict PR3 cleavage sites because we failed to obtain a specific levels similar to that of the commercial IL-33 proteins in mouse PR3 cleavage site by mass spectrometer analysis (data not Raw 264.7 cells (Fig. 7A). Mouse Raw 264.7 cells were treated shown) due to multiband productions in silver staining. 114 266 with various concentrations of mouse IL-33/p1 (Ser –Ile ) We predicted multiple PR3 cleavage sites by a previous for additional cytokine assays. Mouse TNF, MIP-2, and IL-6 report using a highly specific enzymatic activity testing tri-pep- were produced in a dose-dependent manner (Fig. 7B). We tide library against proteinase 3 (38). A single PR3 cleavage site at the N terminus and two cleavage sites at the C terminus were obtained a very similar result of IL-8 production in HMC-1 8208 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 11 •MARCH 9, 2012 Interleukin-33 Processing Enzyme FIGURE 5. Alignment of human and mouse IL-33, prediction of PR3 cleavage sites, and expression of six rIL-33 proteins. A, amino acid sequence of 1 270 1 266 human (Met –Thr ) and mouse (Met –Ile ) was aligned to predict PR3 cleavage sites by consensus sequence. The previously reported caspase cleavage sites are indicated in blue. The predicted PR3 cleavage sites are marked in red. One cleavage site is at the N terminus, and two cleavage sites are at the C terminus. Mature IL-33 from R&D Systems is indicated in green. B, six different rIL-33 proteins were expressed in E. coli as indicated at the top. The human and mouse IL-33 proteins were purified by a Talon and HPLC and then subjected to 10% SDS-PAGE. The purity of each recombinant protein was visualized by silver staining. The data represent one of three independent experiments. ically suppressed by the anti-ST2 antibody (Fig. 8B), and the results were statistically significant in both human and mouse cells assays (Fig. 8, A and B). Time-dependent Activation of Signaling Molecules by IL-33/ p1—Signal transduction of IL-33/p1-activated molecules was also studied. The phosphorylation of IRAK1, NF-B, p38 MAPK, ERK, and JNK was examined with mouse IL-33/p1- treated Raw 264.7 cells because mature IL-33 proteins (Ser – Ile from R&D Systems) are known to activate these inflam- matory signal pathways. The recombinant precursor IL-33 phosphorylated only ERK1/2 at 60 min and remained for 2 h, but the rest of signaling molecules were not phosphorylated FIGURE 6. Biological activities of precursor IL-33 and three new rIL-33 proteins from PR3-cleaved forms. A, biological activities of human IL-33 (20 (Fig. 9A). Contrary to precursor IL-33, mature IL-33 stimula- ng/ml) proteins from three recombinant PR3-cleaved forms, including pro- tion promptly phosphorylated IRAK1 and NF-B in a time-de- IL-33 were examined with human HMC-1 (A) and Raw 264.7 (B) cells. Recom- pendent manner, reached the maximal level at 15 min after binant IL-33/p1 induced cytokines, but the recombinant protein of precursor IL-33, IL-33/p2, and IL-33/p3 were not active. The data represent one of three exposure, and decreased dramatically at 30 min. However, the independent experiments. cont, control. phosphorylation of I-B was delayed and reached a maximum at 60 min. The phosphorylation of p38 MAPK, ERK1/2, and cells, but no additional cytokines were produced in human JNK was similar to that of NF-B; however, its phosphorylation HMC-1 cells (data not shown). was sustained longer than that of IRAK1 and NF-B (Fig. 9B, Anti-ST2 Specifically Inhibits the Cytokine Productions by bottom panels). The phosphorylation pattern of NF-B, p38 IL-33/p1—We examined whether human IL-33 (Ser – 270 114 266 MAPK, ERK1/2, and JNK was similar to IRAK1, although there Thr ) and mouse IL-33/p1 (Ser –Ile ) induce chemokines was a difference in the density of activation. This result sug- and inflammatory cytokines via ST2 on the cell surface of gested the induction of inflammatory cytokines through the HMC-1 and Raw 264.7 cells. These cells were pretreated with activation of IRAK1, NF-B, p38 MAPK, ERK1/2, and JNK sig- anti-ST2 neutralizing antibody along with a control anti- nal pathways. body, and the human and mouse IL-33/p1 proteins were then used to stimulate HMC-1 and Raw 264.7 cells. The DISCUSSION anti-ST2 antibody-pretreated HMC-1 cells produced less IL-8 compared with nontreated cells (Fig. 8A), but the con- In this study, we demonstrate an IL-33-processing enzyme, trol antibody had no effect (data not shown). In mouse Raw which is neutrophil serine proteinase 3 (PR3). PR3 processes 264.7 cells, TNF, MIP-2, and IL-6 productions were specif- chemokine and cytokines such as IL-8 (39), TGF1 (40), mem- MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8209 Interleukin-33 Processing Enzyme FIGURE 7. Comparison of IL-33/p1 with commercial mature IL-33 and a dose-dependent induction of inflammatory cytokines. A, biological activity of human and mouse IL-33/p1 was compared with commercial human and mouse IL-33 proteins (R&D Systems), respectively. Human IL-33/p1 exhibited 80% bioactivity of the commercial human IL-33, whereas the bioactivity of mouse IL-33/p1 was very similar to that of commercial mouse IL-33. B, several concen- trations of IL-33/p1 were treated to mouse Raw 264.7 cells overnight. Inflammatory cytokines (TNF and IL-6) and chemokine (MIP-2) were produced in mouse Raw 264.7 cells in a dose-dependent manner. The data represent one of three independent experiments. FIGURE 8. Human and mouse IL-33/p1 produced cytokines via ST2. HMC-1 cells (A) and Raw 264.7 cells (B) were pretreated with anti-ST2 antibody, and then human and mouse IL-33/p1 proteins were treated to cells. The culture supernatant was harvested, and secreted IL-8 in HMC-1 cells (A) and TNF, MIP-2, and IL-6 in Raw 264.7 cells (B) were measured. The anti-ST2 antibody specifically inhibited both human and mouse IL-33/p1-induced cytokine productions. Mean S.E. of cytokines. **, p  0.01 (n  3 per group). The data represent one of three independent experiments. cont, control. brane-bound TNF (41), IL-1 (42), IL-18 (43), and IL-32 (44) dependent degradation terminated its biological activity after and ameliorate the biological activity of each cytokine. PR3 pro- 30 min of incubation (Fig. 2). The rIL-33 and rIL-18 proteins cesses the precursor form of IL-1 family ligands such as IL-1 were cleaved resulting in productions of multibands (Fig. 3B) and IL-18 (42, 43). The biological activity of PR3-cleaved unlike caspase-1-cleaved precursor IL-18 (Fig. 3A). 117 270 114 mature human IL-33 (Ser –Thr ) and mouse IL-33 (Ser – In addition, PR3 cleaved IL-33 proteins stimulated HMC-1 Ile ) proteins were studied and compared with that of mature cells and induced IL-8 production; however, its activity was 112 270 109 266 human IL-33 (Ser –Thr ) and mouse IL-33 (Ser –Ile ). reduced along with increasing PR3 incubation time. These results suggest that PR3 has dual functions (activation/termi- PR3-cleaved human and mouse mature IL-33 was highly active in producing chemokines and cytokines. nation) in IL-33 biology during infection or inflammation. The The role of caspase-1 in the activation of IL-33 was unclear in time-dependent reduction of IL-33 activity (Fig. 2B) was con- previous studies. The original study of IL-33 suggested that firmed with an alternative method. We verified reduction of 110 111 caspase-1 cleaves the amino acid residue (Asp mature size IL-33 by Western blot (Fig. 4). –Ser )on human precursor IL-33. This result was generated by the incu- In contrast to the first report (6), it has been reported that bation of in vitro-translated IL-33 rather than precursor IL-33 full-length precursor IL-33 is active, and the processing by protein (6). Interestingly, our result in Fig. 3A showed that caspase-1 results in IL-33 inactivation (34). Another report on human and mouse precursor IL-33 proteins were not cleaved IL-33 processing by Luthi et al. (35) demonstrated that apopto- by caspase-1, whereas precursor IL-18 protein was specifically tic caspases process precursor IL-33 in apoptotic cells resulting processed by caspase-1 and became an active mature IL-18 (45). in inactivation of precursor IL-33, whereas precursor IL-33 The precursor IL-33 ligand affinity column isolated urinary released from necrotic cell is spontaneously active. The result PR3 from concentrated human urine (Fig. 1). This result and suggested that the cleavage site of caspase-1 does not occur at 110 111 previous reports of PR3 in the processing of IL-1 (IL-1F2) and the site initially proposed (Asp –Ser ) but rather at amino 178 179 IL-18 (IL-1F4) led us to investigate whether PR3 is an enzyme acid residue Asp –Gly , which is the consensus site of for IL-33 processing. In the presence of PR3, precursor IL-33 cleavage by caspase-3 (34, 35, 46). Apoptotic caspase-3 and 7 gained its biological function at 5 min of incubation, and a time- abolishes the biological activity of precursor IL-33 by cleavage 8210 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 11 •MARCH 9, 2012 Interleukin-33 Processing Enzyme FIGURE 9. Mouse IL-33/p1 induced the phosphorylation of IRAK1, NF-B, I-B, p38 MAPK, p44/42 MAPK, and JNK. Mouse precursor IL-33 (A) and IL-33/p1 (B) (50 ng/ml) were used to treat mouse macrophage Raw 264.7 cells at the indicated time points. A, p44/42 MAPK was only phosphorylated by mouse precursor IL-33 at 60 min, but the rest of the signaling molecules was not affected. B, phosphorylation of IRAK1, NF-B, p38 MAPK, p44/42 MAPK, and JNK was significantly increased at 15 min. IL-33/p1 phosphorylated I-B at 30 min and reached its highest at 60 min. The phosphorylation of p38 MAPK was sustained longer. The bottom of each panel exhibits the expression level of nonphosphorylated signaling molecule in cell lysates to show an equal amount of protein sample was loaded in each lane. The data represent one of three independent experiments. 178 179 of the amino acid residue Asp –Gly . However, these stud- which was produced by PR3 cleavage site at the N terminus, was ies have been performed in vitro; the biological activity of the highly active. Although the predicted caspase-1 cleavage site 110 111 precursor IL-33 was shown by only NF-B luciferase assay (35) (Asp –Ser ) is unclear, commercial mature human IL-33 112 270 109 266 upon transfection of precursor IL-33 cDNA. Unlike the previ- (Ser –Thr ) and mouse IL-33 (Ser –Ile ) are highly ous study, the present experiments were performed with PR3- active in the production of cytokine. Our mature human 117 270 117 270 114 266 cleaved forms of mature human IL-33 (Ser –Thr ) and IL-33/p1 (Ser –Thr ) and mouse IL-33/p1 (Ser –Ile ) 114 266 mouse IL-33 (Ser –Ile ) proteins, inducing inflammatory exhibited potent activity, and the induction of cytokine produc- cytokines and activating the phosphorylation of signaling tion levels were very similar to that of commercial human and molecules. mouse mature IL-33 (Fig. 7A). Another study reported calpain-dependent processing of We produced human and mouse mature rIL-33 according to precursor IL-33 like IL-1, which was exhibited by treatment the consensus amino acid sequence of PR3 cleavage sites in a with calcium ionophore. Calpain processes precursor IL-33, previous report (38). Wysocka et al. (38) described the chemical which produces mature IL-33 in human epithelial and endothe- synthesis of the selective chromogenic/fluorogenic substrates lial cells in the absence of IL-33 activity (47). Contrary to this and deconvolution of the tri-peptide library against proteinase result, Ohno et al. (48) reported that caspase-1/8 and calpain 3 with the general formula ABZ-X X X -ANB-NH , which 3 2 1 2 are dispensable for IL-33 release from macrophage and mast yielded the active sequence. The identification of the predicted cells. Endogenous mouse IL-33 was secreted spontaneously. PR3-cleaved p1, p2, and 3 fragments by enzymatic activity of IL-33 secretion from cells was increased by LPS or phorbol PR3 was extremely difficult because PR3 cleaves pro-IL-33 12-myristate 13-acetate plus ionomycin in the peritoneal transiently and eventually abolishes IL-33 activity by fragmen- macrophages of caspase-1-deficient BALB/c mice (48). The tation (Fig. 3B). As shown in Fig. 4, PR3 digested mature IL-33 discrepancy of these results may be due to species differences. completely at 30 min. PR3-cleaved human and mouse p2 are Although there is significant sequence homology between indicated by arrows (2nd and 4th lanes of Fig. 3B), but p1 and p3 human and mouse IL-33, they share only 55% identity at amino were indistinguishable because p1 migrated as PR3 (far right acid level. The experiment of calpain-dependent IL-33 process- lane of Fig. 3B compared with Western blot in Fig. 4) and p3 ing was performed with human cell lines, but the opposite migrated as pro-IL-33 (2nd lane of Fig. 5B compared with that report by Ohno et al. (48) was carried out using the peritoneal of the 4th lane). macrophage of caspase-1-deficient mice. Although our prediction of the PR3 cleavage site Ile – 117 113 114 PR3-cleaved precursor IL-33 sufficiently induces inflamma- Ser of human IL-33 and Thr –Ser of mouse IL-33 is a tory cytokines, and the biological activity of mature IL-33/p1, highly conserved consensus sequence of a potential PR3 cleav- MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8211 Interleukin-33 Processing Enzyme 14. Brint, E. K., Xu, D., Liu, H., Dunne, A., McKenzie, A. N., O’Neill, L. A., and age site, as indicated by a previous report (38), we cannot Liew, F. Y. 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Csernok, E., Szymkowiak, C. H., Mistry, N., Daha, M. R., Gross, W. L., and 48. Ohno, T., Oboki, K., Kajiwara, N., Morii, E., Aozasa, K., Flavell, R. A., Kekow, J. (1996) Transforming growth factor- (TGF-) expression and Okumura, K., Saito, H., and Nakae, S. (2009) Caspase-1, caspase-8, and interaction with proteinase 3 (PR3) in anti-neutrophil cytoplasmic anti- calpain are dispensable for IL-33 release by macrophages. J. Immunol. 183, body (ANCA)-associated vasculitis. Clin. Exp. Immunol. 105, 104–111 7890–7897 MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8213 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity *

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American Society for Biochemistry and Molecular Biology
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0021-9258
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10.1074/jbc.m111.295055
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 11, pp. 8205–8213, March 9, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Contradictory Functions (Activation/Termination) of Neutrophil Proteinase 3 Enzyme (PR3) in Interleukin-33 Biological Activity Received for publication, August 19, 2011, and in revised form, January 12, 2012 Published, JBC Papers in Press, January 23, 2012, DOI 10.1074/jbc.M111.295055 ‡1 §1 ‡1 ‡ ‡ ‡ ‡ Suyoung Bae , Taebong Kang , Jaewoo Hong , Siyoung Lee , Jida Choi , Hyunjhung Jhun , Areum Kwak , ‡ ‡ ‡ ‡2 Kwangwon Hong , Eunsom Kim , Seunghyun Jo , and Soohyun Kim From the Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701 and the Department of Biotechnology, College of Biomedical and Health Science, Konkuk University, Chung-Ju 380-701, Korea Background: The maturation process of IL-33 (IL-1F11) remains unclear. Results: IL-33 ligand affinity column isolates neutrophil proteinase 3. Conclusion: PR3 is an IL-33-processing enzyme. Significance: PR3 has a dual function in IL-33 biological activity. IL-1 family ligand does not possess a typical hydrophobic type immune responses like immunity against nematodes and signal peptide and needs a processing enzyme for maturation. allergic diseases (2–6), mast cells (4, 7), basophils (8), and eosin- The maturation process of IL-33 (IL-1F11), a new member of ophils (5, 9, 10). IL-33 also induces non-Th2 inflammatory the IL-1 family ligand, remains unclear. Precursor IL-33 cytokines such as TNF, IL-1, or IL-6 (11–13). It has also been ligand affinity column isolates neutrophil proteinase 3 (PR3) suggested that the properties of IL-33 are cytokines or a nuclear from human urinary proteins. PR3 is a known IL-1 family transcription factor like IL-1 and high mobility group protein ligand-processing enzyme for IL-1 (IL-1F2) and IL-18 (IL- B1 (4, 5, 14–23). 1F4), including other inflammatory cytokines. We investi- ST2 (also known as IL-1RL1, DER4, Fit-1, or T1), which was gated PR3 in the maturation process of precursor IL-33 originally discovered as an orphan receptor (24–28), is a ligand because we isolated urinary PR3 by using the precursor IL-33 binding chain of IL-33 (6). IL-33 receptor complex for signaling ligand affinity column. PR3 converted inactive human and is composed of the ligand chain ST2 and signal transducing mouse precursor IL-33 proteins to biological active forms; chain IL-1 receptor accessory protein (IL-1RAcP) (2, 6, 29). however, the increase of PR3 incubation time abrogated This receptor complex activates downstream signaling mole- IL-33 activities. Unlike caspase-1-cleaved precursor IL-18, cules such as NF-B and AP-1 through IL-1 receptor-associ- PR3 cut precursor IL-33 and IL-18 at various sites and yielded ated kinase, TNF receptor-associated factor 6, and/or MAPKs multibands. The increased incubation period of PR3 abated (6). These cells produce inflammatory cytokines and chemo- mature IL-33 in a time-dependent manner. The result is con- kines, including IL-4, IL-5, IL-6, IL-13, and IL-8, by stimulation sistent with the decreased bioactivity of IL-33 along with the of IL-33 (5, 7, 9, 30). Recently, it has been reported that circu- increased PR3 incubation time. Six different human and lating CD34 hematopoietic progenitors expressed ST2 and mouse recombinant IL-33 proteins were expressed by the responded to IL-33 by releasing high levels of Th2-associated predicted consensus amino acid sequence of PR3 cleavage cytokines (31). Such results suggest potential roles of IL-33 in sites and tested for bioactivities. The human IL-33/p1 was Th2-associated immune responses, and IL-33 seems to be highly active, but human IL-33/p2 and p3 proteins were inac- closely related to allergic inflammatory diseases, including tive. Our results suggest the dual functions (activation/termi- asthma and atopic dermatitis. nation) of PR3 in IL-33 biological activity. and IL-18, is produced as a precur- IL-33, similarly to IL-1 sor IL-33 molecule. This precursor IL-33 does not have a signal peptide to be secreted; it is released extracellularly as a mature IL-33 is a new member of the IL-1 family ligand and was protein after cleavage (6). It is known that inflammatory originally discovered as a nuclear factor from high endothelial and IL-18 (32, caspases are necessary for the cleavage of IL-1 venules (1). IL-33 is considered to be critical in inducing Th2- 33). Although it has been suggested that precursor IL-33 is pro- cessed by caspase-1 (6), the exact role of caspases in IL-33 biol- * This work was supported by the National Research Foundation funded by ogy still remains unclear (34, 35). Korean Government Grants WCU R33-2008-000-10022-0 and KRF-2008- In this study, we identified and characterized a processing 313-C00644 and Korea Healthcare Technology R&D Project, Ministry of enzyme of precursor IL-33. PR3 converts inactive precursor Health & Welfare, Republic of Korea Grant A100460. These authors contributed equally to this work. IL-33 to active form, whereas a longer incubation period of PR3 To whom correspondence should be addressed: Laboratory of Cytokine abolishes IL-33 activity. This result suggests the contrasting Immunology, Dept. of Biomedical Science and Technology, Konkuk Uni- functions (activation/termination) of PR3 in precursor IL-33 versity, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701 Korea. Tel.: 82-2- 457-0868; Fax: 82-2-2030-7788; E-mail: [email protected]. bioactivity. MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8205 This is an Open Access article under the CC BY license. Interleukin-33 Processing Enzyme MATERIALS AND METHODS ance liquid chromatography (GE Healthcare) with C4 column (Grace Vydac, Hesperia, CA). The two-step purified recombi- RT-PCR and Molecular Cloning for Escherichia coli Expres- nant proteins were tested for endotoxin level (below 0.25 EU sion Vectors—Total RNA was isolated with TRI Reagent per g of recombinant protein) by using LAL method (Cape (Sigma) from human PC-3 cells and mouse lung tissue. A pair of Cod, East Falmouth, MA) according to the manufacturer’s human IL-33 sense primers 5-ACAGAATACTGAAAAATG- instructions and then used for experiments. AAGCC-3 and reverse primer 5-CTTCTCCAGTGGTAGC- Caspase-1 and PR3 Cleavage Test—Caspase-1 (10 units from ATTTG-3, mouse IL-33 sense primers 5-ATGAGACCTAG- Millipore, Temecula, CA) and the recombinant proteins of pre- AATGAAGTATTC-3 and reverse primer 5-GCACAGGCG- cursor human IL-33, mouse IL-33, and human IL-18 (500 ng) TTTTACTGCATT-3, and -actin sense primers 5-ACCAA- were incubated in 20 l of a reaction buffer (25 mM K HEPES, CTGGGACGACATGGA-3 and reverse primer 5-GTGATG- 1mM DTT, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, pH 7.5) at ACCTGGCCGTCAGG-3 was used for the RT-PCR. Moloney 37 °C for 30 min. After the reaction, the sample was subjected to murine leukemia virus-RT (Beams Bio, Korea) was used for 10% SDS-PAGE for silver staining. converting 2 g of total RNA to first strand cDNA, and then For PR3 cleavage test with different IL-1 family ligands, com- PCR was performed at 94 °C for 45 s, 70 °C for 2 min, and 59 °C mercial PR3 (100 ng from Athens Research & Technology) and for 1 min for 30 cycles. the recombinant proteins of precursor human IL-33, mouse For E. coli expression vector, the PCR product of human and IL-33, and human IL-18 (500 ng) were incubated in 20 lofa mouse precursor IL-33 cDNA was ligated into T&A cloning reaction buffer (50 mM Tris, pH 8) at room temperature. The vector (RBC, Taiwan). We designed the sense primer with an PR3-cleaved samples were subjected to 10% SDS-PAGE for sil- EcoRI (GAATTC) site and the reverse primer with a KpnI ver staining. (GGTACC) site to transfer the amplified PCR product into Western Blots—For the detection of PR3-cleaved human pProEX/HTa (Invitrogen). rIL -33, the samples were loaded on 10% SDS-PAGE. Anti-hu- The insert of human precursor IL-33, IL-33/p2, and IL-33/p3 man IL-33 polyclonal antibody was developed by immunizing was amplified with the same sense primer (5-GAACGAATT- three BALB/c mice (6 weeks old) with mature human IL-33 CATGAAGCCTAAAATGAAG-3). The insert of human pre- 112 270 (Ser –Thr ) protein (data not shown). Human IL-33 poly- cursor IL-33 and IL-33/p1 was amplified with the same reverse clonal antibody was used for detecting a mature size of IL-33, primer (5-GAACGGTACCCTAAGTTTCAGAGAGCTT- which was cleaved by PR3. In addition, an anti-human PR3 3). The sense primer of human IL-33/p1 (5-TAAAGAATTC- monoclonal antibody (36) was obtained from YbdYbiotech ACCTATTACAGAGTATCTT-3) and the reverse primer of (Seoul, Korea) and used for detecting urinary PR3. Peroxidase- IL-33/p2 (5-TATTGGTACCTTAGACAAAGAAGGCCTG- conjugated goat anti-mouse IgG secondary antibody (Jackson 3) and IL-33/p3 (5-TATTGGTACCTTATATAAACACTC- ImmunoResearch) was used to develop the blots by using Supex CAGG-3) were used to amplify cDNA inserts. (Neuronex, Korea) and LAS-4000 imaging device (Fujifilm, For mouse precursor IL-33 and IL-33/p1, the same reverse Japan). primer (5-TATTGGTACCTTAGATTTTCGAGAGCTT-3) For detecting the phosphorylation of signaling molecules and the sense primer of mouse precursor IL-33 (5-TAAAG- (IRAK1, NF-B, p38 MAPK, p44/42 MAPK, and JNK), AATTCATGAGACCTAGAATGAAG-3) and IL-33/p1 (5- HMC-1 and Raw 264.7 cells were stimulated with precursor TAAAGAATTCTCACTTTTAACACAGTCT-3) were used and mature IL-33 at various time points. Cells were lysed to amplify cDNA inserts. The PCR product of a single band with kinase lysis buffer (Cell Signaling Technology, Beverly, from each construct was digested with EcoRI and KpnI and MA) and then subjected to 10% SDS-PAGE. The samples then transferred into pProEX/HTa (Invitrogen) for recombi- were transferred to nitrocellulose membranes. The mem- nant protein expression in E. coli. branes were blocked in 3% BSA/TBST (Santa Cruz Biotech- Isolation of Urinary IL-33 Interacting Molecule from Concen- nology, Santa Cruz, CA). The membranes were probed first trated Human Urine—Human precursor IL-33 (3 mg) was with rabbit anti-phospho-IRAK1, mouse anti-phospho-NF- immobilized by coupling to Affi-Gel-15 beads according to the B, rabbit anti-phospho-p38 MAPK, mouse anti-phospho- manufacturer’s instructions (Bio-Rad). Batches of 300 ml of p44/42 MAPK, or rabbit anti-phospho-JNK (Cell Signaling crude urinary proteins concentrated 500-fold were passed over Technology). The membranes were reprobed with rabbit the precursor IL-33-bound beads at 4 °C. The column was anti-NF-B, p38 MAPK (Santa Cruz Biotechnology), p44/42 washed with 500 ml of phosphate buffer containing 0.5 MAPK, JNK, or IRAK1 (Cell Signaling Technology) for nor- sodium chloride, pH 8. Bound proteins were eluted in a 1-ml malization of each protein and then normalized finally with volume of solution containing 25 mM citric acid, pH 2.2, and goat anti-actin (Santa Cruz Biotechnology). then the eluted fraction was immediately neutralized with 2 M Cell Culture and Bioassay—Mouse macrophage Raw 264.7 glycine. cell line was obtained from American Type Culture Collection Generation of Recombinant Protein—Six recombinant (hu- (ATCC) and maintained according to the instructions. HMC-1 man and mouse precursor IL-33, human and mouse IL-33/p1, cells were cultured in Iscove’s modified Dulbecco’s medium and human IL-33/p2, IL-33/p3) proteins were expressed in enriched with 10% FBS. For a blockade of ST2, a fresh medium E. coli Rosetta cells (Novagen, Madison, WI) and purified by a TALON affinity column (Invitrogen). The TALON affinity-pu- rified recombinant proteins were subjected to high perform- The abbreviation used is: rIL, recombinant IL. 8206 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 11 •MARCH 9, 2012 Interleukin-33 Processing Enzyme FIGURE 1. Isolation of an IL-33 interacting molecule by using ligand affinity column. A, precursor IL-33 affinity chromatography column isolated urinary IL-33-interacting proteins and was visualized by silver-stained 10% SDS-PAGE. Four fractions were eluted from the IL-33 ligand affinity column. Molecular mass is indicated on the left. The arrows indicate the IL-33-binding proteins, 56-, 28-, and minor 36-kDa bands. B, mouse anti-human PR3 antibody was used for verifying urinary PR3 in the fractions from the IL-33 ligand affinity column. The molecular sizes of56 (dimer form) and 28 kDa (monomer form), including the minor 36-kDa (precursor PR3) bands, were detected in the fractions. The data represent one of three independent experiments. (0.2 ml) containing various concentrations of rIL-33 and 0.5 g of anti-ST2 (R&D Systems) was used. Human HMC-1 and mouse Raw 264.7 cells were treated with different stimuli, and the cell culture supernatant was harvested the next day for cyto- kine assay. Measurement of Cytokine Level—Human IL-8, mouse TNF, mouse MIP-2, and mouse IL-6 ELISA kits were obtained from R&D Systems. Cytokine levels were measured in culture super- natants by sandwich ELISA according to manufacturer’s FIGURE 2. PR3 enhancing precursor IL-33-induced cytokine production. instructions. A, PR3-preincubated precursor IL-33 induced IL-8 in a dose-dependent man- Statistical Analysis—The data are expressed as means  S.E. ner. PR3 (25 ng/ml) was preincubated for 5 min with various concentrations of Statistical significance of differences were analyzed by precursor IL-33 as indicated on the bottom and then used for stimulating HMC-1 cells. B, precursor IL-33 (100 ng/ml) activity was gradually decreased unpaired, two-tailed Student’s t test. Values of p  0.05 were along with increasing PR3 incubation times. The data represent one of three considered statistically significant. independent experiments. cont, control. RESULTS and U937), and the molecular size of precursor PR3 is similar Isolation of an IL-33 Interacting Molecule from Urinary to the 36-kDa band (data not shown). Proteins—In an attempt to isolate a soluble IL-33 interacting PR3 Converted Precursor IL-33 into Mature IL-33—The bio- molecule, concentrated urine was applied to a column com- logical activity of human and mouse precursor IL-33 was exam- posed of precursor IL-33 covalently immobilized to agarose ined in the presence of PR3. The pretreatment of PR3 activated beads. After extensive washing with PBS, interacting pro- precursor IL-33 and induced IL-8 production by a dose-depen- teins were eluted at low pH citric acid solution. Aliquots of dent manner, but precursor IL-33 alone did not induce IL-8 the various fractions were resolved by 10% SDS-PAGE, and production (Fig. 2A). The time course of PR3 pretreatment the protein bands were visualized by silver staining. The revealed that PR3 induced the highest activity of IL-33 at 5 min, broad bands corresponding to specific IL-33 interacting and its activity was decreased along with increasing incubation proteins, 56- and 28-kDa bands including the minor times (Fig. 2B). PR3 pretreatment for 30 min completely abol- 36-kDa band, were detected mainly in fractions 2 and 3 (Fig. ished IL-33 activity. 1A). The 56- and 28-kDa protein bands in elution fraction 3 Examination of IL-33 Cleavage by PR3—We tested the cleav- were excised and analyzed by mass spectrometer analysis. age of human and mouse precursor IL-33 proteins with The result of mass spectrometer revealed that the bands caspase-1 and PR3. Caspase-1 specifically cleaved precursor were neutrophil proteinase 3 (PR3). We verified urinary PR3 IL-18 resulting in the production of 18-kDa mature IL-18, in the eluted fractions with a monoclonal antibody specific which was active in the A549R cell line (data not shown) (37), to human PR3. The anti-human PR3 monoclonal antibody but precursor IL-33 proteins were not cleaved by caspase-1 recognized the 56-, 36-, and 28-kDa urinary PR3 and com- (Fig. 3A). Next we examined the cleavage of precursor IL-33 mercial PR3 (Fig. 1B). The results suggest that 28 and 56 kDa proteins with PR3. As shown in Fig. 3B, PR3 cleaved the pre- are the monomer and dimer forms of mature PR3, respec- cursor IL-33 and produced multibands unlike caspase-1- tively. The additional minor 36-kDa band could be the pre- cleaved IL-18 (Fig. 3A). PR3 also cleaved precursor IL-18 as cursor PR3 because the anti-PR3 monoclonal antibody rec- multiband products in silver staining (Fig. 3B, 2nd lane from ognized the precursor PR3 in human monocyte cells (THP-1 right). MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8207 Interleukin-33 Processing Enzyme FIGURE 3. Silver staining of caspase-1 and PR3-cleaved precursor IL-33. A, process of precursor human IL-33 (250 ng/lane), mouse IL-33 (100 ng/lane), and IL-18 (500 ng/lane) was examined with caspase-1 (Casp-1) (10 units/lane). The preincubation of caspase-1 specifically cleaved precursor IL-18 and produced a single band of mature IL-18, molecular mass of 18 kDa, but both human and mouse precursor IL-33 were not affected. B, same amount of recombinant IL-1 family ligands was used for testing if these ligands were processed by PR3 (100 ng/lane). Unlike caspase-1, PR3 cleaved precursor IL-18 and produced multibands in the 2nd lane from right. The pattern of PR3-cleaved precursor IL-33 was very similar in both human and mouse. The data represent one of three independent experiments. predicted by the consensus sequence of the PR3 cleavage site. The predicted PR3 cleavage sites are indicated in red as p1, p2, and p3 (Fig. 5A). The previously reported caspase cleavage sites are shown in blue (Fig. 5A). We expressed six rIL-33 proteins as shown at the top of Fig. 5B. The purified rIL-33 proteins were subjected to 10% SDS-PAGE, and the result showed a single band of correct molecular size of each recombinant protein (Fig. 5B). The upper bands of precursor IL-33 and IL-33/p2–3 are dimer forms that were verified by Western blot (data not shown). Additional experiments tested the biological activity of 112 270 109 mature human IL-33 (Ser –Thr ) and mouse IL-33 (Ser – Ile ) in the presence or absence of the N-terminal His tag. The N-terminal His tag did not affect the bioactivities of mature human and mouse rIL-33 (data not shown). Biological Activity of rIL-33/p1 Proteins—Four human IL-33 proteins (Fig. 5B) were used to test the biological activities with 117 270 FIGURE 4. Time-dependent reduction of a mature size of IL-33. Time human HMC-1 cells. IL-33/p1 (Ser –Thr ) was highly course study of human precursor (Pro) IL-33 (100 ng/lane) cleavage was per- active and induced IL-8 production, but precursor IL-33 formed in the presence of PR3 (20 ng/lane). The precursor IL-33 was promptly 1 270 1 220 (Met –Thr ), huIL-33/p2 (Met –Val ), and huIL-33/p3 reduced after 5 min of PR3 incubation. Western blot revealed that a mature 1 240 (Mat) size of IL-33 was decreased by a time-dependent manner. The data (Met –Ile ) were not active (Fig. 6A). We repeated cytokine represent one of three independent experiments. assay with mouse Raw 264.7 cells, and mouse TNF production was consistent with that of IL-8 (Fig. 6B). We did not produce Time-dependent Reduction of a Mature Size IL-33 by PR3— the mouse form of IL-33/p2 and IL-p3 proteins because both IL-33 protein was incubated at different time points to examine huIL-33/p2 and huIL-33/p3 were not active (Fig. 6). whether the multiband products of the PR3 cleaved is a mature We continued to produce the large scale of human rIL-33/p1 IL-33. We used the anti-IL-33 antibody to detect mature IL-33. 117 270 114 266 (Ser –Thr ) and mouse rIL-33/p1 (Ser –Ile ) because The result of the Western blot exhibited that PR3-cleaved human rIL-33/p1 was only active in the cytokine assay (Fig. 6). mature IL-33 was highest at 5 min and decreased gradually 117 270 Human IL-33/p1 (Ser –Thr ) has six amino acids less than along with incubation time (Fig. 4). This result is consistent 111 270 the originally proposed mature human IL-33 (Ser –Thr ) with bioactivity of the PR3-incubated precursor IL-33 in Fig. by caspase-1 cleavage (6). The biological activity of human 112 270 2B. Mature rIL-33 (Ser –Thr ) was completely degraded by 117 270 114 266 rIL-33 (Ser –Thr ) and mouse rIL-33/p1 (Ser –Ile ) PR3 after 30 min of incubation (Fig. 4, far right lane). was compared with commercial human mature IL-33 (Ser Design and Expression of PR3-cleaved rIL-33 Proteins—We 270 109 266 Thr ) and mouse mature IL-33 (Ser –Ile ) from R&D Sys- aligned the amino acid sequence of human and mouse IL-33 to tems. Our human and mouse IL-33/p1 proteins induced TNF predict PR3 cleavage sites because we failed to obtain a specific levels similar to that of the commercial IL-33 proteins in mouse PR3 cleavage site by mass spectrometer analysis (data not Raw 264.7 cells (Fig. 7A). Mouse Raw 264.7 cells were treated shown) due to multiband productions in silver staining. 114 266 with various concentrations of mouse IL-33/p1 (Ser –Ile ) We predicted multiple PR3 cleavage sites by a previous for additional cytokine assays. Mouse TNF, MIP-2, and IL-6 report using a highly specific enzymatic activity testing tri-pep- were produced in a dose-dependent manner (Fig. 7B). We tide library against proteinase 3 (38). A single PR3 cleavage site at the N terminus and two cleavage sites at the C terminus were obtained a very similar result of IL-8 production in HMC-1 8208 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 11 •MARCH 9, 2012 Interleukin-33 Processing Enzyme FIGURE 5. Alignment of human and mouse IL-33, prediction of PR3 cleavage sites, and expression of six rIL-33 proteins. A, amino acid sequence of 1 270 1 266 human (Met –Thr ) and mouse (Met –Ile ) was aligned to predict PR3 cleavage sites by consensus sequence. The previously reported caspase cleavage sites are indicated in blue. The predicted PR3 cleavage sites are marked in red. One cleavage site is at the N terminus, and two cleavage sites are at the C terminus. Mature IL-33 from R&D Systems is indicated in green. B, six different rIL-33 proteins were expressed in E. coli as indicated at the top. The human and mouse IL-33 proteins were purified by a Talon and HPLC and then subjected to 10% SDS-PAGE. The purity of each recombinant protein was visualized by silver staining. The data represent one of three independent experiments. ically suppressed by the anti-ST2 antibody (Fig. 8B), and the results were statistically significant in both human and mouse cells assays (Fig. 8, A and B). Time-dependent Activation of Signaling Molecules by IL-33/ p1—Signal transduction of IL-33/p1-activated molecules was also studied. The phosphorylation of IRAK1, NF-B, p38 MAPK, ERK, and JNK was examined with mouse IL-33/p1- treated Raw 264.7 cells because mature IL-33 proteins (Ser – Ile from R&D Systems) are known to activate these inflam- matory signal pathways. The recombinant precursor IL-33 phosphorylated only ERK1/2 at 60 min and remained for 2 h, but the rest of signaling molecules were not phosphorylated FIGURE 6. Biological activities of precursor IL-33 and three new rIL-33 proteins from PR3-cleaved forms. A, biological activities of human IL-33 (20 (Fig. 9A). Contrary to precursor IL-33, mature IL-33 stimula- ng/ml) proteins from three recombinant PR3-cleaved forms, including pro- tion promptly phosphorylated IRAK1 and NF-B in a time-de- IL-33 were examined with human HMC-1 (A) and Raw 264.7 (B) cells. Recom- pendent manner, reached the maximal level at 15 min after binant IL-33/p1 induced cytokines, but the recombinant protein of precursor IL-33, IL-33/p2, and IL-33/p3 were not active. The data represent one of three exposure, and decreased dramatically at 30 min. However, the independent experiments. cont, control. phosphorylation of I-B was delayed and reached a maximum at 60 min. The phosphorylation of p38 MAPK, ERK1/2, and cells, but no additional cytokines were produced in human JNK was similar to that of NF-B; however, its phosphorylation HMC-1 cells (data not shown). was sustained longer than that of IRAK1 and NF-B (Fig. 9B, Anti-ST2 Specifically Inhibits the Cytokine Productions by bottom panels). The phosphorylation pattern of NF-B, p38 IL-33/p1—We examined whether human IL-33 (Ser – 270 114 266 MAPK, ERK1/2, and JNK was similar to IRAK1, although there Thr ) and mouse IL-33/p1 (Ser –Ile ) induce chemokines was a difference in the density of activation. This result sug- and inflammatory cytokines via ST2 on the cell surface of gested the induction of inflammatory cytokines through the HMC-1 and Raw 264.7 cells. These cells were pretreated with activation of IRAK1, NF-B, p38 MAPK, ERK1/2, and JNK sig- anti-ST2 neutralizing antibody along with a control anti- nal pathways. body, and the human and mouse IL-33/p1 proteins were then used to stimulate HMC-1 and Raw 264.7 cells. The DISCUSSION anti-ST2 antibody-pretreated HMC-1 cells produced less IL-8 compared with nontreated cells (Fig. 8A), but the con- In this study, we demonstrate an IL-33-processing enzyme, trol antibody had no effect (data not shown). In mouse Raw which is neutrophil serine proteinase 3 (PR3). PR3 processes 264.7 cells, TNF, MIP-2, and IL-6 productions were specif- chemokine and cytokines such as IL-8 (39), TGF1 (40), mem- MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8209 Interleukin-33 Processing Enzyme FIGURE 7. Comparison of IL-33/p1 with commercial mature IL-33 and a dose-dependent induction of inflammatory cytokines. A, biological activity of human and mouse IL-33/p1 was compared with commercial human and mouse IL-33 proteins (R&D Systems), respectively. Human IL-33/p1 exhibited 80% bioactivity of the commercial human IL-33, whereas the bioactivity of mouse IL-33/p1 was very similar to that of commercial mouse IL-33. B, several concen- trations of IL-33/p1 were treated to mouse Raw 264.7 cells overnight. Inflammatory cytokines (TNF and IL-6) and chemokine (MIP-2) were produced in mouse Raw 264.7 cells in a dose-dependent manner. The data represent one of three independent experiments. FIGURE 8. Human and mouse IL-33/p1 produced cytokines via ST2. HMC-1 cells (A) and Raw 264.7 cells (B) were pretreated with anti-ST2 antibody, and then human and mouse IL-33/p1 proteins were treated to cells. The culture supernatant was harvested, and secreted IL-8 in HMC-1 cells (A) and TNF, MIP-2, and IL-6 in Raw 264.7 cells (B) were measured. The anti-ST2 antibody specifically inhibited both human and mouse IL-33/p1-induced cytokine productions. Mean S.E. of cytokines. **, p  0.01 (n  3 per group). The data represent one of three independent experiments. cont, control. brane-bound TNF (41), IL-1 (42), IL-18 (43), and IL-32 (44) dependent degradation terminated its biological activity after and ameliorate the biological activity of each cytokine. PR3 pro- 30 min of incubation (Fig. 2). The rIL-33 and rIL-18 proteins cesses the precursor form of IL-1 family ligands such as IL-1 were cleaved resulting in productions of multibands (Fig. 3B) and IL-18 (42, 43). The biological activity of PR3-cleaved unlike caspase-1-cleaved precursor IL-18 (Fig. 3A). 117 270 114 mature human IL-33 (Ser –Thr ) and mouse IL-33 (Ser – In addition, PR3 cleaved IL-33 proteins stimulated HMC-1 Ile ) proteins were studied and compared with that of mature cells and induced IL-8 production; however, its activity was 112 270 109 266 human IL-33 (Ser –Thr ) and mouse IL-33 (Ser –Ile ). reduced along with increasing PR3 incubation time. These results suggest that PR3 has dual functions (activation/termi- PR3-cleaved human and mouse mature IL-33 was highly active in producing chemokines and cytokines. nation) in IL-33 biology during infection or inflammation. The The role of caspase-1 in the activation of IL-33 was unclear in time-dependent reduction of IL-33 activity (Fig. 2B) was con- previous studies. The original study of IL-33 suggested that firmed with an alternative method. We verified reduction of 110 111 caspase-1 cleaves the amino acid residue (Asp mature size IL-33 by Western blot (Fig. 4). –Ser )on human precursor IL-33. This result was generated by the incu- In contrast to the first report (6), it has been reported that bation of in vitro-translated IL-33 rather than precursor IL-33 full-length precursor IL-33 is active, and the processing by protein (6). Interestingly, our result in Fig. 3A showed that caspase-1 results in IL-33 inactivation (34). Another report on human and mouse precursor IL-33 proteins were not cleaved IL-33 processing by Luthi et al. (35) demonstrated that apopto- by caspase-1, whereas precursor IL-18 protein was specifically tic caspases process precursor IL-33 in apoptotic cells resulting processed by caspase-1 and became an active mature IL-18 (45). in inactivation of precursor IL-33, whereas precursor IL-33 The precursor IL-33 ligand affinity column isolated urinary released from necrotic cell is spontaneously active. The result PR3 from concentrated human urine (Fig. 1). This result and suggested that the cleavage site of caspase-1 does not occur at 110 111 previous reports of PR3 in the processing of IL-1 (IL-1F2) and the site initially proposed (Asp –Ser ) but rather at amino 178 179 IL-18 (IL-1F4) led us to investigate whether PR3 is an enzyme acid residue Asp –Gly , which is the consensus site of for IL-33 processing. In the presence of PR3, precursor IL-33 cleavage by caspase-3 (34, 35, 46). Apoptotic caspase-3 and 7 gained its biological function at 5 min of incubation, and a time- abolishes the biological activity of precursor IL-33 by cleavage 8210 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 11 •MARCH 9, 2012 Interleukin-33 Processing Enzyme FIGURE 9. Mouse IL-33/p1 induced the phosphorylation of IRAK1, NF-B, I-B, p38 MAPK, p44/42 MAPK, and JNK. Mouse precursor IL-33 (A) and IL-33/p1 (B) (50 ng/ml) were used to treat mouse macrophage Raw 264.7 cells at the indicated time points. A, p44/42 MAPK was only phosphorylated by mouse precursor IL-33 at 60 min, but the rest of the signaling molecules was not affected. B, phosphorylation of IRAK1, NF-B, p38 MAPK, p44/42 MAPK, and JNK was significantly increased at 15 min. IL-33/p1 phosphorylated I-B at 30 min and reached its highest at 60 min. The phosphorylation of p38 MAPK was sustained longer. The bottom of each panel exhibits the expression level of nonphosphorylated signaling molecule in cell lysates to show an equal amount of protein sample was loaded in each lane. The data represent one of three independent experiments. 178 179 of the amino acid residue Asp –Gly . However, these stud- which was produced by PR3 cleavage site at the N terminus, was ies have been performed in vitro; the biological activity of the highly active. Although the predicted caspase-1 cleavage site 110 111 precursor IL-33 was shown by only NF-B luciferase assay (35) (Asp –Ser ) is unclear, commercial mature human IL-33 112 270 109 266 upon transfection of precursor IL-33 cDNA. Unlike the previ- (Ser –Thr ) and mouse IL-33 (Ser –Ile ) are highly ous study, the present experiments were performed with PR3- active in the production of cytokine. Our mature human 117 270 117 270 114 266 cleaved forms of mature human IL-33 (Ser –Thr ) and IL-33/p1 (Ser –Thr ) and mouse IL-33/p1 (Ser –Ile ) 114 266 mouse IL-33 (Ser –Ile ) proteins, inducing inflammatory exhibited potent activity, and the induction of cytokine produc- cytokines and activating the phosphorylation of signaling tion levels were very similar to that of commercial human and molecules. mouse mature IL-33 (Fig. 7A). Another study reported calpain-dependent processing of We produced human and mouse mature rIL-33 according to precursor IL-33 like IL-1, which was exhibited by treatment the consensus amino acid sequence of PR3 cleavage sites in a with calcium ionophore. Calpain processes precursor IL-33, previous report (38). Wysocka et al. (38) described the chemical which produces mature IL-33 in human epithelial and endothe- synthesis of the selective chromogenic/fluorogenic substrates lial cells in the absence of IL-33 activity (47). Contrary to this and deconvolution of the tri-peptide library against proteinase result, Ohno et al. (48) reported that caspase-1/8 and calpain 3 with the general formula ABZ-X X X -ANB-NH , which 3 2 1 2 are dispensable for IL-33 release from macrophage and mast yielded the active sequence. The identification of the predicted cells. Endogenous mouse IL-33 was secreted spontaneously. PR3-cleaved p1, p2, and 3 fragments by enzymatic activity of IL-33 secretion from cells was increased by LPS or phorbol PR3 was extremely difficult because PR3 cleaves pro-IL-33 12-myristate 13-acetate plus ionomycin in the peritoneal transiently and eventually abolishes IL-33 activity by fragmen- macrophages of caspase-1-deficient BALB/c mice (48). The tation (Fig. 3B). As shown in Fig. 4, PR3 digested mature IL-33 discrepancy of these results may be due to species differences. completely at 30 min. PR3-cleaved human and mouse p2 are Although there is significant sequence homology between indicated by arrows (2nd and 4th lanes of Fig. 3B), but p1 and p3 human and mouse IL-33, they share only 55% identity at amino were indistinguishable because p1 migrated as PR3 (far right acid level. The experiment of calpain-dependent IL-33 process- lane of Fig. 3B compared with Western blot in Fig. 4) and p3 ing was performed with human cell lines, but the opposite migrated as pro-IL-33 (2nd lane of Fig. 5B compared with that report by Ohno et al. (48) was carried out using the peritoneal of the 4th lane). macrophage of caspase-1-deficient mice. Although our prediction of the PR3 cleavage site Ile – 117 113 114 PR3-cleaved precursor IL-33 sufficiently induces inflamma- Ser of human IL-33 and Thr –Ser of mouse IL-33 is a tory cytokines, and the biological activity of mature IL-33/p1, highly conserved consensus sequence of a potential PR3 cleav- MARCH 9, 2012• VOLUME 287 • NUMBER 11 JOURNAL OF BIOLOGICAL CHEMISTRY 8211 Interleukin-33 Processing Enzyme 14. Brint, E. K., Xu, D., Liu, H., Dunne, A., McKenzie, A. N., O’Neill, L. A., and age site, as indicated by a previous report (38), we cannot Liew, F. Y. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Mar 9, 2012

Keywords: Caspase; Cytokine; Inflammation; Interleukin; Signal Transduction

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