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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
Suyoung Bae
‡1
, Taebong Kang
§1
, Jaewoo Hong
‡1
, Siyoung Lee
‡
, Jida Choi
‡
, Hyunjhung Jhun
‡
, Areum Kwak
‡
,
Kwangwon Hong
‡
, Eunsom Kim
‡
, Seunghyun Jo
‡
, and Soohyun Kim
‡2
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
signal peptide and needs a processing enzyme for maturation.
The maturation process of IL-33 (IL-1F11), a new member of
the IL-1 family ligand, remains unclear. Precursor IL-33
ligand affinity column isolates neutrophil proteinase 3 (PR3)
from human urinary proteins. PR3 is a known IL-1 family
ligand-processing enzyme for IL-1
!
(IL-1F2) and IL-18 (IL-
1F4), including other inflammatory cytokines. We investi-
gated PR3 in the maturation process of precursor IL-33
because we isolated urinary PR3 by using the precursor IL-33
ligand affinity column. PR3 converted inactive human and
mouse precursor IL-33 proteins to biological active forms;
however, the increase of PR3 incubation time abrogated
IL-33 activities. Unlike caspase-1-cleaved precursor IL-18,
PR3 cut precursor IL-33 and IL-18 at various sites and yielded
multibands. The increased incubation period of PR3 abated
mature IL-33 in a time-dependent manner. The result is con-
sistent with the decreased bioactivity of IL-33 along with the
increased PR3 incubation time. Six different human and
mouse recombinant IL-33 proteins were expressed by the
predicted consensus amino acid sequence of PR3 cleavage
sites and tested for bioactivities. The human IL-33/p1 was
highly active, but human IL-33/p2 and p3 proteins were inac-
tive. Our results suggest the dual functions (activation/termi-
nation) of PR3 in IL-33 biological activity.
IL-33 is a new member of the IL-1 family ligand and was
originally discovered as a nuclear factor from high endothelial
venules (1). IL-33 is considered to be critical in inducing Th2-
type immune responses like immunity against nematodes and
allergic diseases (2– 6), mast cells (4, 7), basophils (8), and eosin-
ophils (5, 9, 10). IL-33 also induces non-Th2 inflammatory
cytokines such as TNF
!
, IL-1
"
, or IL-6 (11–13). It has also been
suggested that the properties of IL-33 are cytokines or a nuclear
transcription factor like IL-1
"
and high mobility group protein
B1 (4, 5, 14–23).
ST2 (also known as IL-1RL1, DER4, Fit-1, or T1), which was
originally discovered as an orphan receptor (24 –28), is a ligand
binding chain of IL-33 (6). IL-33 receptor complex for signaling
is composed of the ligand chain ST2 and signal transducing
chain IL-1 receptor accessory protein (IL-1RAcP) (2, 6, 29).
This receptor complex activates downstream signaling mole-
cules such as NF-
#
B and AP-1 through IL-1 receptor-associ-
ated kinase, TNF receptor-associated factor 6, and/or MAPKs
(6). These cells produce inflammatory cytokines and chemo-
kines, including IL-4, IL-5, IL-6, IL-13, and IL-8, by stimulation
of IL-33 (5, 7, 9, 30). Recently, it has been reported that circu-
lating CD34
!
hematopoietic progenitors expressed ST2 and
responded to IL-33 by releasing high levels of Th2-associated
cytokines (31). Such results suggest potential roles of IL-33 in
Th2-associated immune responses, and IL-33 seems to be
closely related to allergic inflammatory diseases, including
asthma and atopic dermatitis.
IL-33, similarly to IL-1
"
and IL-18, is produced as a precur-
sor IL-33 molecule. This precursor IL-33 does not have a signal
peptide to be secreted; it is released extracellularly as a mature
protein after cleavage (6). It is known that inflammatory
caspases are necessary for the cleavage of IL-1
"
and IL-18 (32,
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-
ogy still remains unclear (34, 35).
In this study, we identified and characterized a processing
enzyme of precursor IL-33. PR3 converts inactive precursor
IL-33 to active form, whereas a longer incubation period of PR3
abolishes IL-33 activity. This result suggests the contrasting
functions (activation/termination) of PR3 in precursor IL-33
bioactivity.
* This work was supported by the National Research Foundation funded by
Korean Government Grants WCU R33-2008-000-10022-0 and KRF-2008-
313-C00644 and Korea Healthcare Technology R&D Project, Ministry of
Health & Welfare, Republic of Korea Grant A100460.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed: Laboratory of Cytokine
Immunology, Dept. of Biomedical Science and Technology, Konkuk Uni-
versity, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701 Korea. Tel.: 82-2-
457-0868; Fax: 82-2-2030-7788; E-mail: soohyun@konkuk.ac.kr.
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.
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MATERIALS AND METHODS
RT-PCR and Molecular Cloning for Escherichia coli Expres-
sion Vectors—Total RNA was isolated with TRI Reagent!
(Sigma) from human PC-3 cells and mouse lung tissue. A pair of
human IL-33 sense primers 5"-ACAGAATACTGAAAAATG-
AAGCC-3" and reverse primer 5"-CTTCTCCAGTGGTAGC-
ATTTG-3", mouse IL-33 sense primers 5"-ATGAGACCTAG-
AATGAAGTATTC-3" and reverse primer 5"-GCACAGGCG-
TTTTACTGCATT-3", and
"
-actin sense primers 5"-ACCAA-
CTGGGACGACATGGA-3" and reverse primer 5"-GTGATG-
ACCTGGCCGTCAGG-3" was used for the RT-PCR. Moloney
murine leukemia virus-RT (Beams Bio, Korea) was used for
converting 2
$
g of total RNA to first strand cDNA, and then
PCR was performed at 94 °C for 45 s, 70 °C for 2 min, and 59 °C
for 1 min for 30 cycles.
For E. coli expression vector, the PCR product of human and
mouse precursor IL-33 cDNA was ligated into T&A cloning
vector (RBC, Taiwan). We designed the sense primer with an
EcoRI (GAATTC) site and the reverse primer with a KpnI
(GGTACC) site to transfer the amplified PCR product into
pProEX/HTa (Invitrogen).
The insert of human precursor IL-33, IL-33/p2, and IL-33/p3
was amplified with the same sense primer (5"-GAACGAATT-
CATGAAGCCTAAAATGAAG-3"). The insert of human pre-
cursor IL-33 and IL-33/p1 was amplified with the same reverse
primer (5"-GAACGGTACCCTAAGTTTCAGAGAGCTT-
3"). The sense primer of human IL-33/p1 (5"-TAAAGAATTC-
ACCTATTACAGAGTATCTT-3") and the reverse primer of
IL-33/p2 (5"-TATTGGTACCTTAGACAAAGAAGGCCTG-
3") and IL-33/p3 (5"-TATTGGTACCTTATATAAACACTC-
CAGG-3") were used to amplify cDNA inserts.
For mouse precursor IL-33 and IL-33/p1, the same reverse
primer (5"-TATTGGTACCTTAGATTTTCGAGAGCTT-3")
and the sense primer of mouse precursor IL-33 (5"-TAAAG-
AATTCATGAGACCTAGAATGAAG-3")andIL-33/p1(5"-
TAAAGAATTCTCACTTTTAACACAGTCT-3") were used
to amplify cDNA inserts. The PCR product of a single band
from each construct was digested with EcoRI and KpnI and
then transferred into pProEX/HTa (Invitrogen) for recombi-
nant protein expression in E. coli.
Isolation of Urinary IL-33 Interacting Molecule from Concen-
trated Human Urine—Human precursor IL-33 (3 mg) was
immobilized by coupling to Affi-Gel-15 beads according to the
manufacturer’s instructions (Bio-Rad). Batches of 300 ml of
crude urinary proteins concentrated 500-fold were passed over
the precursor IL-33-bound beads at 4 °C. The column was
washed with 500 ml of phosphate buffer containing 0.5
M
sodium chloride, pH 8. Bound proteins were eluted in a 1-ml
volume of solution containing 25 m
M citric acid, pH 2.2, and
then the eluted fraction was immediately neutralized with 2
M
glycine.
Generation of Recombinant Protein—Six recombinant (hu-
man and mouse precursor IL-33, human and mouse IL-33/p1,
and human IL-33/p2, IL-33/p3) proteins were expressed in
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-
ance liquid chromatography (GE Healthcare) with C4 column
(Grace Vydac, Hesperia, CA). The two-step purified recombi-
nant proteins were tested for endotoxin level (below 0.25 EU
per
$
g of recombinant protein) by using LAL method (Cape
Cod, East Falmouth, MA) according to the manufacturer’s
instructions and then used for experiments.
Caspase-1 and PR3 Cleavage Test—Caspase-1 (10 units from
Millipore, Temecula, CA) and the recombinant proteins of pre-
cursor human IL-33, mouse IL-33, and human IL-18 (500 ng)
were incubated in 20
$
l of a reaction buffer (25 mM K
!
HEPES,
1m
M DTT, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, pH 7.5) at
37 °C for 30 min. After the reaction, the sample was subjected to
10% SDS-PAGE for silver staining.
For PR3 cleavage test with different IL-1 family ligands, com-
mercial PR3 (100 ng from Athens Research & Technology) and
the recombinant proteins of precursor human IL-33, mouse
IL-33, and human IL-18 (500 ng) were incubated in 20
$
l of a
reaction buffer (50 m
M Tris, pH 8) at room temperature. The
PR3-cleaved samples were subjected to 10% SDS-PAGE for sil-
ver staining.
Western Blots—For the detection of PR3-cleaved human
rIL
3
-33, the samples were loaded on 10% SDS-PAGE. Anti-hu-
man IL-33 polyclonal antibody was developed by immunizing
three BALB/c mice (6 weeks old) with mature human IL-33
(Ser
112
–Thr
270
) protein (data not shown). Human IL-33 poly
-
clonal antibody was used for detecting a mature size of IL-33,
which was cleaved by PR3. In addition, an anti-human PR3
monoclonal antibody (36) was obtained from YbdYbiotech
(Seoul, Korea) and used for detecting urinary PR3. Peroxidase-
conjugated goat anti-mouse IgG secondary antibody (Jackson
ImmunoResearch) was used to develop the blots by using Supex
(Neuronex, Korea) and LAS-4000 imaging device (Fujifilm,
Japan).
For detecting the phosphorylation of signaling molecules
(IRAK1, NF-
#
B, p38 MAPK, p44/42 MAPK, and JNK),
HMC-1 and Raw 264.7 cells were stimulated with precursor
and mature IL-33 at various time points. Cells were lysed
with kinase lysis buffer (Cell Signaling Technology, Beverly,
MA) and then subjected to 10% SDS-PAGE. The samples
were transferred to nitrocellulose membranes. The mem-
branes were blocked in 3% BSA/TBST (Santa Cruz Biotech-
nology, Santa Cruz, CA). The membranes were probed first
with rabbit anti-phospho-IRAK1, mouse anti-phospho-NF-
#
B, rabbit anti-phospho-p38 MAPK, mouse anti-phospho-
p44/42 MAPK, or rabbit anti-phospho-JNK (Cell Signaling
Technology). The membranes were reprobed with rabbit
anti-NF-
#
B, p38 MAPK (Santa Cruz Biotechnology), p44/42
MAPK, JNK, or IRAK1 (Cell Signaling Technology) for nor-
malization of each protein and then normalized finally with
goat anti-actin (Santa Cruz Biotechnology).
Cell Culture and Bioassay—Mouse macrophage Raw 264.7
cell line was obtained from American Type Culture Collection
(ATCC) and maintained according to the instructions. HMC-1
cells were cultured in Iscove’s modified Dulbecco’s medium
enriched with 10% FBS. For a blockade of ST2, a fresh medium
3
The abbreviation used is: rIL, recombinant IL.
Interleukin-33 Processing Enzyme
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(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
instructions.
Statistical Analysis—The data are expressed as means # S.E.
Statistical significance of differences were analyzed by
unpaired, two-tailed Student’s t test. Values of p $ 0.05 were
considered statistically significant.
RESULTS
Isolation of an IL-33 Interacting Molecule from Urinary
Proteins—In an attempt to isolate a soluble IL-33 interacting
molecule, concentrated urine was applied to a column com-
posed of precursor IL-33 covalently immobilized to agarose
beads. After extensive washing with PBS, interacting pro-
teins were eluted at low pH citric acid solution. Aliquots of
the various fractions were resolved by 10% SDS-PAGE, and
the protein bands were visualized by silver staining. The
broad bands corresponding to specific IL-33 interacting
proteins, %56- and 28-kDa bands including the minor
36-kDa band, were detected mainly in fractions 2 and 3 (Fig.
1A). The 56- and 28-kDa protein bands in elution fraction 3
were excised and analyzed by mass spectrometer analysis.
The result of mass spectrometer revealed that the bands
were neutrophil proteinase 3 (PR3). We verified urinary PR3
in the eluted fractions with a monoclonal antibody specific
to human PR3. The anti-human PR3 monoclonal antibody
recognized the 56-, 36-, and 28-kDa urinary PR3 and com-
mercial PR3 (Fig. 1B). The results suggest that 28 and 56 kDa
are the monomer and dimer forms of mature PR3, respec-
tively. The additional minor 36-kDa band could be the pre-
cursor PR3 because the anti-PR3 monoclonal antibody rec-
ognized the precursor PR3 in human monocyte cells (THP-1
and U937), and the molecular size of precursor PR3 is similar
to the 36-kDa band (data not shown).
PR3 Converted Precursor IL-33 into Mature IL-33—The bio-
logical activity of human and mouse precursor IL-33 was exam-
ined in the presence of PR3. The pretreatment of PR3 activated
precursor IL-33 and induced IL-8 production by a dose-depen-
dent manner, but precursor IL-33 alone did not induce IL-8
production (Fig. 2A). The time course of PR3 pretreatment
revealed that PR3 induced the highest activity of IL-33 at 5 min,
and its activity was decreased along with increasing incubation
times (Fig. 2B). PR3 pretreatment for 30 min completely abol-
ished IL-33 activity.
Examination of IL-33 Cleavage by PR3—We tested the cleav-
age of human and mouse precursor IL-33 proteins with
caspase-1 and PR3. Caspase-1 specifically cleaved precursor
IL-18 resulting in the production of 18-kDa mature IL-18,
which was active in the A549R
"
cell line (data not shown) (37),
but precursor IL-33 proteins were not cleaved by caspase-1
(Fig. 3A). Next we examined the cleavage of precursor IL-33
proteins with PR3. As shown in Fig. 3B, PR3 cleaved the pre-
cursor IL-33 and produced multibands unlike caspase-1-
cleaved IL-18 (Fig. 3A). PR3 also cleaved precursor IL-18 as
multiband products in silver staining (Fig. 3B, 2nd lane from
right).
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 of %56 (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.
FIGURE 2. PR3 enhancing precursor IL-33-induced cytokine production.
A, PR3-preincubated precursor IL-33 induced IL-8 in a dose-dependent man-
ner. PR3 (25 ng/ml) was preincubated for 5 min with various concentrations of
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
along with increasing PR3 incubation times. The data represent one of three
independent experiments. cont, control.
Interleukin-33 Processing Enzyme
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Time-dependent Reduction of a Mature Size IL-33 by PR3—
IL-33 protein was incubated at different time points to examine
whether the multiband products of the PR3 cleaved is a mature
IL-33. We used the anti-IL-33 antibody to detect mature IL-33.
The result of the Western blot exhibited that PR3-cleaved
mature IL-33 was highest at 5 min and decreased gradually
along with incubation time (Fig. 4). This result is consistent
with bioactivity of the PR3-incubated precursor IL-33 in Fig.
2B. Mature rIL-33 (Ser
112
–Thr
270
) was completely degraded by
PR3 after 30 min of incubation (Fig. 4, far right lane).
Design and Expression of PR3-cleaved rIL-33 Proteins—We
aligned the amino acid sequence of human and mouse IL-33 to
predict PR3 cleavage sites because we failed to obtain a specific
PR3 cleavage site by mass spectrometer analysis (data not
shown) due to multiband productions in silver staining.
We predicted multiple PR3 cleavage sites by a previous
report using a highly specific enzymatic activity testing tri-pep-
tide library against proteinase 3 (38). A single PR3 cleavage site
at the N terminus and two cleavage sites at the C terminus were
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
mature human IL-33 (Ser
112
–Thr
270
) and mouse IL-33 (Ser
109
–
Ile
266
) 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
human HMC-1 cells. IL-33/p1 (Ser
117
–Thr
270
) was highly
active and induced IL-8 production, but precursor IL-33
(Met
1
–Thr
270
), huIL-33/p2 (Met
1
–Val
220
), and huIL-33/p3
(Met
1
–Ile
240
) were not active (Fig. 6
A). We repeated cytokine
assay with mouse Raw 264.7 cells, and mouse TNF
!
production
was consistent with that of IL-8 (Fig. 6B). We did not produce
the mouse form of IL-33/p2 and IL-p3 proteins because both
huIL-33/p2 and huIL-33/p3 were not active (Fig. 6).
We continued to produce the large scale of human rIL-33/p1
(Ser
117
–Thr
270
) and mouse rIL-33/p1 (Ser
114
–Ile
266
) because
human rIL-33/p1 was only active in the cytokine assay (Fig. 6).
Human IL-33/p1 (Ser
117
–Thr
270
) has six amino acids less than
the originally proposed mature human IL-33 (Ser
111
–Thr
270
)
by caspase-1 cleavage (6). The biological activity of human
rIL-33 (Ser
117
–Thr
270
) and mouse rIL-33/p1 (Ser
114
–Ile
266
)
was compared with commercial human mature IL-33 (Ser
112
–
Thr
270
) and mouse mature IL-33 (Ser
109
–Ile
266
) from R&D Sys
-
tems. Our human and mouse IL-33/p1 proteins induced TNF
!
levels similar to that of the commercial IL-33 proteins in mouse
Raw 264.7 cells (Fig. 7A). Mouse Raw 264.7 cells were treated
with various concentrations of mouse IL-33/p1 (Ser
114
–Ile
266
)
for additional cytokine assays. Mouse TNF
!
, MIP-2, and IL-6
were produced in a dose-dependent manner (Fig. 7B). We
obtained a very similar result of IL-8 production in HMC-1
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.
FIGURE 4. Time-dependent reduction of a mature size of IL-33. Time
course study of human precursor (Pro) IL-33 (100 ng/lane) cleavage was per-
formed in the presence of PR3 (20 ng/lane). The precursor IL-33 was promptly
reduced after 5 min of PR3 incubation. Western blot revealed that a mature
(Mat) size of IL-33 was decreased by a time-dependent manner. The data
represent one of three independent experiments.
Interleukin-33 Processing Enzyme
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cells, but no additional cytokines were produced in human
HMC-1 cells (data not shown).
Anti-ST2 Specifically Inhibits the Cytokine Productions by
IL-33/p1—We examined whether human IL-33 (Ser
117
–
Thr
270
) and mouse IL-33/p1 (Ser
114
–Ile
266
) induce chemokines
and inflammatory cytokines via ST2 on the cell surface of
HMC-1 and Raw 264.7 cells. These cells were pretreated with
anti-ST2 neutralizing antibody along with a control anti-
body, and the human and mouse IL-33/p1 proteins were
then used to stimulate HMC-1 and Raw 264.7 cells. The
anti-ST2 antibody-pretreated HMC-1 cells produced less
IL-8 compared with nontreated cells (Fig. 8A), but the con-
trol antibody had no effect (data not shown). In mouse Raw
264.7 cells, TNF
!
,MIP-2,andIL-6productionswerespecif-
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
109
–
Ile
266
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
(Fig. 9A). Contrary to precursor IL-33, mature IL-33 stimula-
tion promptly phosphorylated IRAK1 and NF-
#
B in a time-de-
pendent manner, reached the maximal level at 15 min after
exposure, and decreased dramatically at 30 min. However, the
phosphorylation of I-
#
B was delayed and reached a maximum
at 60 min. The phosphorylation of p38 MAPK, ERK1/2, and
JNK was similar to that of NF-
#
B; however, its phosphorylation
was sustained longer than that of IRAK1 and NF-
#
B (Fig. 9B,
bottom panels). The phosphorylation pattern of NF-
#
B, p38
MAPK, ERK1/2, and JNK was similar to IRAK1, although there
was a difference in the density of activation. This result sug-
gested the induction of inflammatory cytokines through the
activation of IRAK1, NF-
#
B, p38 MAPK, ERK1/2, and JNK sig-
nal pathways.
DISCUSSION
In this study, we demonstrate an IL-33-processing enzyme,
which is neutrophil serine proteinase 3 (PR3). PR3 processes
chemokine and cytokines such as IL-8 (39), TGF
"
1 (40), mem-
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
human (Met
1
–Thr
270
) and mouse (Met
1
–Ile
266
) 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.
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
ng/ml) proteins from three recombinant PR3-cleaved forms, including pro-
IL-33 were examined with human HMC-1 (A) and Raw 264.7 (B) cells. Recom-
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
independent experiments. cont, control.
Interleukin-33 Processing Enzyme
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brane-bound TNF
!
(41), IL-1
"
(42), IL-18 (43), and IL-32 (44)
and ameliorate the biological activity of each cytokine. PR3 pro-
cesses the precursor form of IL-1 family ligands such as IL-1
"
and IL-18 (42, 43). The biological activity of PR3-cleaved
mature human IL-33 (Ser
117
–Thr
270
) and mouse IL-33 (Ser
114
–
Ile
266
) proteins were studied and compared with that of mature
human IL-33 (Ser
112
–Thr
270
) and mouse IL-33 (Ser
109
–Ile
266
).
PR3-cleaved human and mouse mature IL-33 was highly active
in producing chemokines and cytokines.
The role of caspase-1 in the activation of IL-33 was unclear in
previous studies. The original study of IL-33 suggested that
caspase-1 cleaves the amino acid residue (Asp
110
–Ser
111
) on
human precursor IL-33. This result was generated by the incu-
bation of in vitro-translated IL-33 rather than precursor IL-33
protein (6). Interestingly, our result in Fig. 3A showed that
human and mouse precursor IL-33 proteins were not cleaved
by caspase-1, whereas precursor IL-18 protein was specifically
processed by caspase-1 and became an active mature IL-18 (45).
The precursor IL-33 ligand affinity column isolated urinary
PR3 from concentrated human urine (Fig. 1). This result and
previous reports of PR3 in the processing of IL-1
"
(IL-1F2) and
IL-18 (IL-1F4) led us to investigate whether PR3 is an enzyme
for IL-33 processing. In the presence of PR3, precursor IL-33
gained its biological function at 5 min of incubation, and a time-
dependent degradation terminated its biological activity after
30 min of incubation (Fig. 2). The rIL-33 and rIL-18 proteins
were cleaved resulting in productions of multibands (Fig. 3B)
unlike caspase-1-cleaved precursor IL-18 (Fig. 3A).
In addition, PR3 cleaved IL-33 proteins stimulated HMC-1
cells and induced IL-8 production; however, its activity was
reduced along with increasing PR3 incubation time. These
results suggest that PR3 has dual functions (activation/termi-
nation) in IL-33 biology during infection or inflammation. The
time-dependent reduction of IL-33 activity (Fig. 2B) was con-
firmed with an alternative method. We verified reduction of
mature size IL-33 by Western blot (Fig. 4).
In contrast to the first report (6), it has been reported that
full-length precursor IL-33 is active, and the processing by
caspase-1 results in IL-33 inactivation (34). Another report on
IL-33 processing by Luthi et al. (35) demonstrated that apopto-
tic caspases process precursor IL-33 in apoptotic cells resulting
in inactivation of precursor IL-33, whereas precursor IL-33
released from necrotic cell is spontaneously active. The result
suggested that the cleavage site of caspase-1 does not occur at
the site initially proposed (Asp
110
–Ser
111
) but rather at amino
acid residue Asp
178
–Gly
179
, which is the consensus site of
cleavage by caspase-3 (34, 35, 46). Apoptotic caspase-3 and &7
abolishes the biological activity of precursor IL-33 by cleavage
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.
Interleukin-33 Processing Enzyme
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of the amino acid residue Asp
178
–Gly
179
. However, these stud
-
ies have been performed in vitro; the biological activity of the
precursor IL-33 was shown by only NF-
#
B luciferase assay (35)
upon transfection of precursor IL-33 cDNA. Unlike the previ-
ous study, the present experiments were performed with PR3-
cleaved forms of mature human IL-33 (Ser
117
–Thr
270
) and
mouse IL-33 (Ser
114
–Ile
266
) proteins, inducing inflammatory
cytokines and activating the phosphorylation of signaling
molecules.
Another study reported calpain-dependent processing of
precursor IL-33 like IL-1
!
, which was exhibited by treatment
with calcium ionophore. Calpain processes precursor IL-33,
which produces mature IL-33 in human epithelial and endothe-
lial cells in the absence of IL-33 activity (47). Contrary to this
result, Ohno et al. (48) reported that caspase-1/8 and calpain
are dispensable for IL-33 release from macrophage and mast
cells. Endogenous mouse IL-33 was secreted spontaneously.
IL-33 secretion from cells was increased by LPS or phorbol
12-myristate 13-acetate plus ionomycin in the peritoneal
macrophages of caspase-1-deficient BALB/c mice (48). The
discrepancy of these results may be due to species differences.
Although there is significant sequence homology between
human and mouse IL-33, they share only 55% identity at amino
acid level. The experiment of calpain-dependent IL-33 process-
ing was performed with human cell lines, but the opposite
report by Ohno et al. (48) was carried out using the peritoneal
macrophage of caspase-1-deficient mice.
PR3-cleaved precursor IL-33 sufficiently induces inflamma-
tory cytokines, and the biological activity of mature IL-33/p1,
which was produced by PR3 cleavage site at the N terminus, was
highly active. Although the predicted caspase-1 cleavage site
(Asp
110
–Ser
111
) is unclear, commercial mature human IL-33
(Ser
112
–Thr
270
) and mouse IL-33 (Ser
109
–Ile
266
) are highly
active in the production of cytokine. Our mature human
IL-33/p1 (Ser
117
–Thr
270
) and mouse IL-33/p1 (Ser
114
–Ile
266
)
exhibited potent activity, and the induction of cytokine produc-
tion levels were very similar to that of commercial human and
mouse mature IL-33 (Fig. 7A).
We produced human and mouse mature rIL-33 according to
the consensus amino acid sequence of PR3 cleavage sites in a
previous report (38). Wysocka et al. (38) described the chemical
synthesis of the selective chromogenic/fluorogenic substrates
and deconvolution of the tri-peptide library against proteinase
3 with the general formula ABZ-X
3
X
2
X
1
-ANB-NH
2
, which
yielded the active sequence. The identification of the predicted
PR3-cleaved p1, p2, and 3 fragments by enzymatic activity of
PR3 was extremely difficult because PR3 cleaves pro-IL-33
transiently and eventually abolishes IL-33 activity by fragmen-
tation (Fig. 3B). As shown in Fig. 4, PR3 digested mature IL-33
completely at 30 min. PR3-cleaved human and mouse p2 are
indicated by arrows (2nd and 4th lanes of Fig. 3B), but p1 and p3
were indistinguishable because p1 migrated as PR3 (far right
lane of Fig. 3B compared with Western blot in Fig. 4) and p3
migrated as pro-IL-33 (2nd lane of Fig. 5B compared with that
of the 4th lane).
Although our prediction of the PR3 cleavage site Ile
116
–
Ser
117
of human IL-33 and Thr
113
–Ser
114
of mouse IL-33 is a
highly conserved consensus sequence of a potential PR3 cleav-
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.
Interleukin-33 Processing Enzyme
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age site, as indicated by a previous report (38), we cannot
exclude alternative PR3 cleavage sites of human IL-33 (Ser
111
–
Ser
112
) and of mouse IL-33 (Leu
108
–Ser
109
). The alternative
PR3 cleavage site was used for producing human and mouse
mature rIL-33 (R&D Systems).
Collectively, we identified PR3 as a processing enzyme of
IL-33. In this study, we proposed PR3 cleavage sites and
expressed various forms of rIL-33 proteins to verify the contrast
functions (activation/termination) of PR3 in the processing of
IL-33 during infection or inflammation. Further in vivo studies
on PR3-induced IL-33 processing by neutrophils or monocytes
could help understand inflammatory disorders of epithelial tis-
sues such as asthma and atopic dermatitis.
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