The Journal of Immunology
Inflammatory Cytokines IL-32 and IL-17 Have Common
Signaling Intermediates despite Differential Dependence on
Emily Turner-Brannen,* Ka-Yee Grace Choi,* Ryan Arsenault,†Hani El-Gabalawy,*
Scott Napper,†and Neeloffer Mookherjee*
Cytokines IL-32 and IL-17 are emerging as critical players in the pathophysiology of immune-mediated chronic inflammatory
diseases. It has been speculated that the molecular mechanisms governing IL-32– and IL-17–mediated cellular responses are
differentially dependent on the TNF pathway. In this study, kinome analysis demonstrated that following stimulation with
cytokine IL-32, but not IL-17, there was increased phosphorylation of a peptide target corresponding to TNF-R1. Consistent
with this observation, blocking TNF-R1 resulted in a suppression of IL-32–induced downstream responses, indicating that IL-32–
mediated activity may be dependent on TNF-R1. In contrast, blocking TNF-R1 did not affect IL-17–induced downstream
responses. Kinome analysis also implicated p300 (transcriptional coactivator) and death-associated protein kinase-1 (DAPK-1)
as signaling intermediates for both IL-32 and IL-17. Phosphorylation of p300 and DAPK-1 upon stimulation with either IL-32 or
IL-17 was confirmed by immunoblots. The presence of common targets was supported by results demonstrating similar down-
stream responses induced in the presence of IL-32 and IL-17, such as transcriptional responses and the direct activation of NF-kB.
Furthermore, knockdown of p300 and DAPK-1 altered downstream responses induced by IL-32 and IL-17, and impacted certain
cellular responses induced by TNF-a and IL-1b. We hypothesize that p300 and DAPK-1 represent nodes where the inflammatory
networks of IL-32 and IL-17 overlap, and that these proteins would affect both TNF-R1–dependent and –independent pathways.
Therefore, p300 and DAPK-1 are viable potential therapeutic targets for chronic inflammatory diseases.
nology, 2011, 186: 7127–7135.
certain cancers. A major limitation in the development of effective
new strategies for the treatment of chronic inflammatory diseases
is an inadequate understanding of the complex molecular mech-
anisms underpinning these disorders. It is well established that
dysregulation of the inflammatory cascade is a major contributing
factor to the development of chronic inflammation. Even though
critical roles of certain proinflammatory cytokines have been well
established in the pathogenesis of chronic inflammatory disorders
(1, 2), the genetic regulation and cellular responses mediated by
inflammatory cytokines appear to be heterogenous (3). This het-
erogeneity may be due to the induction of different disease-
associated pathways in different patients, or it may reflect differ-
The Journal of Immu-
wide range of disorders are characterized by chronic
inflammation, including rheumatoid arthritis (RA), in-
flammatory bowel disease (IBD), psoriasis, asthma, and
ent stages of the disease, or both (4). Heterogeneity in molecular
mechanisms is further reinforced by the fact that response to
pharmacologic treatment varies considerably among patients. For
example, in the case of RA and IBD, up to one third of patients
who receive therapies such as TNF blockers do not respond to
treatment (5). Furthermore, residual inflammatory activity is often
present even among “good” clinical responders of the treatment
(6), and progression of chronic inflammation cannot be com-
pletely controlled by biologic therapies that target proinflam-
matory cytokines such as TNF and IL-1b (5, 6). Improved under-
standing of the molecular processes involved in the various in-
flammatory networks will facilitate identification of overlapping
nodes or common protein targets, which may serve as alternative
therapeutic targets for chronic inflammatory diseases.
Two recently discovered proinflammatory cytokines, IL-32
and IL-17, are associated with the pathogenesis of chronic in-
flammatory diseases (7, 8). IL-32 (NK transcript 4), found in
activated T cells, NK cells, and monocytic cells, is a potent in-
ducer of proinflammatory mediators in diseases such as RA,
atopic dermatitis, and chronic obstructive pulmonary disease (9–
11). IL-32 levels are significantly elevated in RA synovial tissues
and can induce joint inflammation, cartilage damage (9), and os-
teoclast differentiation (12). IL-17, which is primarily synthesized
by T lymphocytes, is a potent inducer of TNF-a, IL-1b, and
matrix metalloproteinases and contributes to the pathogenesis of
chronic inflammation through mechanisms that overlap consider-
ably with IL-32, in the case of RA, ultimately leading to articular
damage (13, 14). Recently, it has been demonstrated that IL-17
can also be secreted by innate immune cells such as macrophages,
dendritic cells, and NK cells (15). The role of IL-17 in chronic
destructive joint inflammation in RA has made it an attractive
*Department of Internal Medicine, Manitoba Centre for Proteomics and Systems
Biology, University of Manitoba, Winnipeg, Manitoba R3E3P4, Canada; and
†Vaccine and Infectious Diseases Organization, Saskatoon, Saskatchewan, Canada
Received for publication July 9, 2010. Accepted for publication April 7, 2011.
This work was supported by funding from the Manitoba Health Research Council and
the Health Sciences Centre Foundation, Winnipeg, Manitoba, Canada.
The kinome data presented in this article have been submitted to the National Center
for Biotechnology Information Gene Expression Omnibus database under accession
Address correspondence and reprint requests to Dr. Neeloffer Mookherjee, Univer-
sity of Manitoba, 799 JBRC, 715 McDermot Avenue, Winnipeg, Manitoba R3E3P4,
Canada. E-mail address: email@example.com
Abbreviations used in this article: DAPK-1, death-associated protein kinase-1; FLS,
fibroblast-like synoviocyte; IBD, inflammatory bowel disease; NSC, nonsilencing
control; qRT-PCR, quantitative real-time PCR; RA, rheumatoid arthritis; siRNA,
small interfering RNA; TC, tissue culture.
therapeutic target, and inhibition of this molecule and its down-
stream effectors is now in the advanced stages of clinical testing
One important difference between IL-32 and IL-17 is that these
cytokines may be differentially dependent on the TNF pathway.
The role of IL-32 in inflammation may, in part, involve TNF-
dependent mechanisms (17), whereas the function of IL-17 in
the sustenance and escalation of inflammation in arthritic con-
ditions may be, in part, TNF independent (13). We hypothesized
that a comparative evaluation of cellular responses induced in the
presence of IL-32 and IL-17 would likely permit identification of
common molecular targets of these cytokines, which may play
a part in both TNF-dependent and -independent processes. This
study investigated cellular responses induced by IL-32 and IL-17
in macrophages and fibroblast-like synoviocytes (FLS), two crit-
ical cell types contributing to the disease process in chronic in-
flammatory arthritis, such as that seen in RA. Kinome analysis and
subsequent immunoblots demonstrated that transcriptional coac-
tivator p300 (EP300), as well as the death-associated protein
kinase-1 (DAPK-1), had increased levels of phosphorylation in
response to either IL-32 or IL-17 stimulation. Moreover, knock-
down of these proteins altered downstream responses induced by
IL-32 and IL-17, and changed certain responses induced in the
presence of TNF-a and IL-1b. We suggest that the identified
common protein targets p300 and DAPK-1 may be involved in the
inflammatory networks for both TNF-dependent and -indepen-
dent processes, and therefore are potential therapeutic targets for
chronic inflammatory diseases.
Materials and Methods
Cell culture and isolation
HumanFLS wereisolatedfromsynovialtissues obtainedfrom patientswith
osteoarthritis who had given informed consent (in accordance with a pro-
tocol by the Institutional Review Board at the University of Manitoba,
Winnipeg, MB, Canada). Briefly, the tissues were digested with 1 mg/ml
collagenase and 0.05 mg/ml hyaluronidase (both obtained from Sigma-
Aldrich, Oakville, ON, Canada) in HBSS (Life Technologies, Invitrogen
Canada, Burlington, ON, Canada) for 2–3 h at 37˚C. The cells were cul-
tured in DMEM media containing 1 mM L-glutamine (Life Technologies)
supplemented with 1 mM sodium pyruvate and 0.1 mM nonessential
amino acids (referred to as complete DMEM henceforth), containing 10%
(v/v) FBS in a humidified incubator at 37˚C and 10% CO2. A rabbit
synoviocyte cell line, HIG-82 (ATCC CRL-1832), was cultured in Ham’s
F-12 growth medium containing 1 mM L-glutamine (Life Technologies)
supplemented with sodium pyruvate (referred to as complete F-12 media
henceforth), containing 10% (v/v) FBS in a humidified incubator at 37˚C
and 5% CO2. Confluent HIG-82 or human FLS were trypsinized with 1:3
dilution of 0.5% trypsin-EDTA (Invitrogen) in HBSS. The synoviocytes
(either HIG-82 or human FLS) were seeded at 2 3 104cells/ml, either 1 ml
per well in 24-well tissue culture (TC) plates, 0.5 ml per well in 48-well
TC plates, or 3 ml per well in 6-well TC plates, as required. The syno-
viocytes were propagated overnight in their respective complete media
containing 10% (v/v) FBS. The culture media were changed the next day
to complete media containing 1% (v/v) FBS before addition of the various
Human monocytic THP-1 (ATCC TIB-202) cells were cultured in RPMI
1640 media containing 2 mM L-glutamine and 1 mM sodium pyruvate
(referred to as complete RPMI media henceforth), supplemented with 10%
(v/v) FBS, and maintained in a humidified incubator at 37˚C and 5% CO2.
The THP-1 cells were differentiated to plastic-adherent macrophage-like
cells by treatment with PMA (Sigma-Aldrich Canada), as previously de-
scribed (18). Cellular cytotoxicity was evaluated after 24-h stimulation
with the various stimulants for all cell types used in this study by moni-
toring the release of lactate dehydrogenase with a colorimetric detection
kit (Roche Diagnostics, Laval, QC, Canada).
Stimulants, reagents, and Abs
Recombinant human cytokines TNF-a, IL-1b, IL-17A/F (referred to as IL-
17 hereafter), and IL-32g (referred to as IL-32 hereafter) were all obtained
from eBioscience (San Diego, CA). Bacterial LPS from E. coli was
obtained from Sigma-Aldrich. Anti-human mAb directed against TNF-R1
(MAB625) and p300 Ab was obtained from R&D Systems, (Minneapolis,
MN). Ab directed to phospho-p300 (Ser1834) and mAb to total DAPK-1
were obtained from MJS Biolynx (Brockville, ON, Canada). mAb directed
against phospho–DAPK-1 (Ser308) was obtained from Santa Cruz Bio-
technology, (Santa Cruz, CA). Anti-HDAC1 polyclonal Ab was obtained
from Thermo Scientific (Pierce Biotechnology, Rockford, IL). mAb to
GAPDH, Abs to human NF-kB subunits p50 and p65, and HRP-linked
purified anti-rabbit IgG and anti-mouse IgG secondary Abs were all
obtained from Cell Signaling Technology, distributed by New England
Biolabs (Pickering, ON, Canada). Cycloheximide was obtained from EMD
chemicals (Newark, NJ).
TC supernatants were centrifuged at 1500 3 g for 5–7 min to obtain cell-
free samples, and aliquots were stored at 220˚C until further use. Pro-
duction of chemokine Gro-a was monitored in the TC supernatants by
ELISA employing human Gro-a DuoSet (R&D Systems) per the manu-
facturer’s instructions. Production of cytokine TNF-a was monitored in the
TC supernatants, using specific Ab pairs from eBioscience, and production
of IL-8 was monitored using specific Ab pairs from R&D Systems,
according to the manufacturer’s instructions. The concentration of cyto-
kines or chemokines in the TC supernatants was evaluated by establishing
a standard curve with serial dilutions of the recombinant human cytokines
Human monocytic THP-1 cells were differentiated to plastic-adherent
macrophage-like cells, as described above, and the cells were rested for
24 h. Following that, the media were changed to RPMI complete media
containing 1% (v/v) FBS, and the cells were stimulated with either IL-32
(20 ng/ml) or IL-17 (20 ng/ml) for 15 min. Subsequently, cellular lysates
were prepared in lysis buffer containing 20 mM Tris-HCl with pH 7.5, 150
mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium fluoride, 1 mM
sodium orthovanadate, 2.5 mM sodium pyrophosphate, protease inhibitor
mixture (Sigma-Aldrich), and 1% (v/v) Triton X-100, as previously de-
scribed (19). An array of peptides representing 300 selected phosphory-
lation events were incubated with the cellular lysates for quantifying
global kinase activity, as explained previously (19). Patterns of differen-
tial phosphorylation of the peptides upon stimulation with IL-32 and
IL-17 were comprehensively analyzed after normalization and background
correction of signal strength, as noted earlier (19).
Total cell lysates were prepared in lysis buffer containing 20 mM Tris-
HCl with pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium
fluoride, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate,
lysates were electrophoretically resolved on 4–12% NuPAGE Bis-Tris gels
(Invitrogen,), followed by transfer to nitrocellulose membranes (Millipore
Canada). The membranes were subsequently blocked with TBST con-
taining 5% skimmed milk powder, and probed with various Abs, as in-
dicated, in TBST containing 3% skimmed milk powder. Affinity purified
HRP-linked secondary Abs were used for detection, as required. The
membranes were developed with the Amersham ECL detection system
(GE Healthcare, Baie d’Urfe, QC, Canada) according to the manufacturer’s
TNF-R blocking assay
Human monocytic THP-1 cells were differentiated to plastic-adherent
macrophage-like cells by treatment with PMA for 24 h (Sigma-Aldrich),
as described above. The cells were rested for an additional 24 h in com-
changed to complete RPMI containing 1% (v/v) FBS. Neutralization or
blocking of TNF-R1 was performed using a specific mAb, MAB625 (R&D
Systems), as previously described (20). Briefly, the cells were preincubated
with the MAB625 for TNF-R1 (20 mg/ml) for 1 h, followed by treatment
with the various stimulants, as indicated. RNA was isolated from these
cells after 4 h of stimulation for the evaluation of transcriptional responses
of chemokine CXCL-1 (Gro-a) and proinflammatory cytokine IL-23
employing quantitative real-time PCR (qRT-PCR). The TC supernatants
were monitored for chemokine Gro-a and IL-8 production after 24 h of
stimulation by ELISA.
7128COMMON SIGNALING INTERMEDIATES OF IL-32 AND IL-17
Human FLS or plastic-adherent THP-1 macrophage-like cells were stim-
ulated with various stimulants, as indicated, for 4 h. RNAwas isolated and
subsequently analyzed for gene expression by qRT-PCR, using SuperScript
III Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen),
according to the manufacturer’s instructions, in the ABI Prism 7000 se-
quence detection system (Applied Biosystems). Fold changes were cal-
culated using the comparative Ct method (21), after normalization with
18S RNA primers. The primers are listed in Table II.
Translocation of NF-kB subunits p50 and p65
Nuclear extracts were prepared using NE-PER extraction reagents (Thermo
Fisher Scientific), according to the manufacturer’s instructions. To monitor
direct NF-kB activation, equivalent nuclear extracts (5–8 mg) were re-
solved on 4–12% NuPAGE Bis-Tris gels (Invitrogen) and transferred to
nitrocellulose membranes. The membranes were blocked with TBST
containing 3% fish skin gelatin (Sigma-Aldrich) and probed with Abs
specific for either NF-kB subunit p50 or p65, in TBST containing 1% fish
gelatin. The membranes were also probed with Ab to human histone
protein HADC-1 to assess the equivalent protein loading. Affinity purified
HRP-linked secondary Abs were used for detection, and the membranes
were developed with the ECL detection system.
NF-kB activation assay
To monitor direct NF-kB activation in synoviocytes, a rabbit synoviocyte
cell line, HIG-82 (ATCC CRL-1832), was transiently transfected with
pNFkB-MetLuc2-Reporter Vector (Clontech Laboratories, Mountain
View, CA) or the provided control vector, per the manufacturer’s instruc-
tions. Various stimulants were added to the transfected cells in culture
media containing 1% (v/v) FBS. The cells were stimulated with either
recombinant human IL-32 or IL-17, and in parallel with known activators
of NF-kB, such as proinflammatory recombinant human cytokines TNF-a
and IL-1b, for 4 or 6 h. These time points were selected according to the
manufacturer’s recommendations. The activation of NF-kB was monitored
by employing the Ready-To-Glow Secreted NF-kB Luciferase Reporter
Assay (Clontech), per the manufacturer’s directions.
Gene silencing using small interfering RNA
HumanmonocyticTHP-1cells were treatedwith1 mMAccell SMARTpool
small interfering RNA (siRNA) for either human p300 (EP300), human
DAPK-1, or nonsilencing control (NSC) in Accell delivery media (Dhar-
macon, Thermo Fisher Scientific), per the manufacturer’s instructions, for
96 h. Subsequently, the cells were differentiated by PMA treatment (as
described above) using complete RPMI media containing 10% (v/v) FBS
and rested for 24 h before stimulation. The plastic-adherent macrophage-
like THP-1 cells were stimulated with either IL-32 (20 ng/ml), IL-17 (20
ng/ml), TNF-a (10 ng/ml), IL-1b (10 ng/ml), or bacterial LPS (10 ng/ml)
for 24 h. TC supernatants were monitored for chemokine IL-8 and cyto-
kine TNF-a production by ELISA.
IL-32– and IL-17–induced protein production in human
macrophages and FLS
Cytokines IL-32 and IL-17 induce the production of proin-
flammatory cytokines such as TNF-a and chemokines in macro-
phages and synovial fibroblasts under inflammatory conditions
(16, 22). In this study, plastic-adherent macrophage-like THP-1
cells (in vitro) were stimulated with either IL-17 or IL-32 (5–100
ng/ml), and the TC supernatants were monitored for cytokines
TNF-a and IL-1b and for production of chemokines Gro-a and
IL-8 by ELISA. Production of both TNF-a and IL-1b following
stimulation with IL-32 from 20 ng/ml onward was dose dependent
and significant (p , 0.05) (Fig. 1A). IL-32 also induced significant
(p , 0.01) production of chemokines Gro-a and IL-8 from 5 to
100 ng/ml (Fig. 1B). Similarly, production of IL-8 upon stimula-
tion with 10, 20, or 50 ng/ml of IL-17 was significant (p , 0.05),
but the amount of IL-8 produced decreased at 100 ng/ml (Fig. 1C).
Taken together, these results indicated that both cytokine IL-32
and IL-17 induced downstream protein production in macrophage-
like THP-1 cells, and that there was significant (p , 0.05) protein
production upon stimulation with either 20 or 50 ng/ml of the
cytokines for all the read-outs monitored in this study. We further
evaluated protein production in synovial fibroblasts. Human FLS
were stimulated with either IL-32 or IL-17 (10 or 20 ng/ml), and
the TC supernatants were monitored for TNF-a, IL-1b, and Gro-a
IL-32 (5–100 ng/ml) for 24 h. TC supernatants were monitored for the production of inflammatory cytokines TNF-a and IL-1b (A) and chemokines Gro-a
and IL-8 (B) by ELISA. C, Macrophage-like THP-1 cells were stimulated with IL-17 (5–100 ng/ml) for 24 h, and the TC supernatants were monitored for
the production of IL-8 by ELISA. Results are shown after subtraction of background levels of cytokines or chemokines monitored in unstimulated cells. D,
Human FLS isolated from synovial tissues (ex vivo) were stimulated with either IL-32 or IL-17 (10 or 20 ng/ml) for 24 h. The TC supernatants were
monitored for chemokine Gro-a production by ELISA. All results are an average of four independent experiments (for FLS isolated from four independent
donors) 6 SE. *p , 0.05, **p , 0.01.
Protein production in the presence of IL-17 and IL-32. Plastic-adherent human macrophage-like THP-1 cells (in vitro) were stimulated with
The Journal of Immunology 7129
production. Gro-a production upon IL-17 stimulation in human
FLS was robust, whereas IL-32 did not produce significant che-
mokine production (Fig. 1D). In this study, human FLS did not
produce significant amounts of TNF-a or IL-1b following stim-
ulation with either IL-17 or IL-32 (data not shown). On the basis
of these results, macrophage-like THP-1 cells were selected for
a comparative analysis of induced cellular responses upon stim-
ulation with IL-32 and IL-17 at 20 ng/ml.
Protein phosphorylations induced in the presence of IL-32 and
Phosphorylation of proteins is a critical mechanism in the regu-
lation of cellular processes. This process is meticulously regulated
by enzymes known as kinases, which are increasingly being
identified as drug targets for a variety of diseases (23, 24). We
interrogated kinase activities (kinome) induced in the presence of
the cytokines IL-32 and IL-17, using peptide arrays representing
300 peptides, printed in triplicate, representing selected phos-
phorylation events, as previously described (19). Because both IL-
32 and IL-17 at 20 ng/ml induced significant (p , 0.05) protein
production in human macrophage-like THP-1 cells (Fig. 1), these
cells were used for the comparative kinome analysis. Macrophage-
like THP-1 cells were stimulated with either IL-32 (20 ng/ml) or
IL-17 (20 ng/ml) for 15 min, and the peptide arrays were used to
comprehensively analyze protein phosphorylation profiles in the
presence of these cytokines, as previously explained (19). The
phosphorylations of the peptides on the array were quantified in
the cytokine-treated cells relative to the unstimulated control cells.
Differentially phosphorylated targets were defined as $1.5-fold
increase or decrease (p , 0.05) in phosphorylation, compared
with unstimulated control cells. The kinome data have been de-
posited to National Center for Biotechnology Information’s Gene
Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/
query/acc.cgi) under series accession number GSE28649. Two key
observations from the kinome analysis were 1) IL-32 significantly
induced the phosphorylation of TNF-R1 by more than 4-fold,
whereas the state of TNF-R1 phosphorylation was not altered in
the presence of IL-17 when compared with the unstimulated con-
trol cells (Table I); and 2) two proteins, p300 and DAPK-1, were
significantly phosphorylated in the presence of either IL-32 or
IL-17 (Table I), indicating that these proteins are common phos-
phorylation targets of these cytokines.
Subsequent probing of immunoblots with specific Abs to human
phospho-p300 (Ser1834) and phospho–DAPK-1 (Ser308) conclu-
sively demonstrated that stimulation of human macrophage-like
THP-1 cells with either IL-32 or IL-17 resulted in the increased
phosphorylation of both p300 and DAPK-1 when compared with
unstimulated control cells after 15 min (Fig. 2).
Alteration of IL-32–induced responses on blocking of TNF-R1
Kinome analysis showed that IL-32 induced the phosphorylation of
TNF-R1 (4.7-fold increase, p , 0.01), whereas the phosphoryla-
tion state of TNF-R1 after stimulation with IL-17 was not altered,
compared with that in unstimulated cells (Table I). This obser-
vation was in agreement with previous studies hypothesizing that
IL-32–mediated pathogenesis in chronic inflammatory diseases
may be in part dependent on TNF-a (17). Therefore we in-
vestigated the role of TNF-R1 in IL-32–induced downstream
responses by blocking receptor activity, using a specific neutral-
izing mAb, as previously described (20). Human macrophage-like
THP-1 cells were pretreated with mAb MAB625 specific for TNF-
R1 (20 mg/ml) for 1 h, followed by stimulation with either IL-32
(20 ng/ml), IL-17 (20 ng/ml), TNF-a (10 ng/ml), or IL-1b (10 ng/
ml) for 4 or 24 h. Transcriptional responses were evaluated by
qRT-PCR (primers used shown in Table II) after 4 h of stimula-
tion. Gene expression of Gro-a (Fig. 3A) and IL-23 (Fig. 3B)
induced in the presence of IL-32 and TNF-a was significantly
(p , 0.05) suppressed, by .70%, in the presence of the neu-
tralizing mAb to TNF-R1. In contrast, transcriptional responses
induced in the presence of either IL-17 or IL-1b were not altered
(Fig. 3A, 3B). A similar trend was also observed at the protein
level; blocking TNF-R1 significantly (p , 0.01) suppressed IL-
32–induced chemokine Gro-a production by 35 6 5% and TNF-
a–induced Gro-a production by 60% after 24 h of stimulation
(Fig. 3C). No difference in IL-1b–induced Gro-a production was
observed (Fig. 3C). Blocking TNF-R1 significantly (p , 0.05)
suppressed IL-32–induced IL-8 production, by .85% (Fig. 3D);
however, it did not alter the production of TNF-a–induced IL-8
production (Fig. 3D).
Common downstream responses induced in the presence of
cytokines IL-32 and IL-17
We showed that both IL-32 and IL-17 induced the phosphorylation
of two common proteins (Table I, Fig. 2). However, phosphory-
lation of the common proteins p300 and DAPK-1 was not abro-
gated upon stimulation with either IL-32 or IL-17 in the presence
of cycloheximide, an inhibitor of protein biosynthesis (data not
shown). This finding indicates that these common signaling events
may be indirect. Nevertheless, as the presence of common sig-
naling intermediates would result in similar downstream cellular
responses, we further interrogated transcriptional responses and
activation of the key transcription factor NF-kB in the presence of
IL-32 and IL-17 in macrophages and human FLS. The cells were
stimulated with either IL-32 or IL-17, and transcriptional re-
sponses were monitored after 4 h of stimulation by qRT-PCR.
Both IL-32 and IL-17 induced significant (p , 0.05) gene ex-
pression of TNF-a, IL-23, and Gro-a, between 2- and 12-fold
higher than that in unstimulated control cells in macrophages
(Fig. 4A). Even though a modest—i.e., ∼2-fold change—increase
in gene expression was produced upon stimulation with IL-17,
compared with that in unstimulated control cells, this increase
was consistent and statistically significant (p , 0.05) in
macrophage-like THP-1 cells. Similarly, a significant increase in
TNF-a, IL-1b, IL-6, and IL-8 gene expression was observed in
Table I. IL-32– and IL-17–induced protein phosphorylations in human monocytic cells
Phosphoprotein IDKinase Target
Fold Changep ,
Fold Changep ,
Human macrophage-like THP-1 cells were stimulated with either IL-32 or IL-17 (20 ng/ml) for 15 min. A kinome screen
using peptide arrays representing 300 phosphorylation targets of kinase activity was employed for quantifying kinase activity.
Differentially phosphorylated targets were defined as $1.5-fold increase or decrease (p , 0.05) in phosphorylation compared
with that in unstimulated control cells.
7130COMMON SIGNALING INTERMEDIATES OF IL-32 AND IL-17
human FLS (ex vivo) upon stimulation with either IL-17 or IL-32
Activation of the key inflammatory transcription factor NF-kB
was evaluated by monitoring the nuclear translocation of NF-kB
subunits p50 and p65 following stimulation with IL-32 and IL-17.
Although NF-kB has five different subunits, inflammatory regu-
lation is most commonly implicated by the functions of subunits
p50 and p65. Thus, human FLS and macrophage-like THP-1 cells
were stimulated with cytokines—either IL-32, IL-17, TNF-a, or
IL-1b—for 30 min, and the nuclear extracts were probed with Abs
specific to NF-kB subunits p50 and p65 by Western blots. In-
creased nuclear localization of NF-kB subunit p50 was demon-
strated following stimulation with either IL-32 or IL-17 in both
human FLS (Fig. 5A) or macrophage-like THP-1 cells (Fig. 5B),
and these responses were similar to that seen after stimulation
with inflammatory cytokines TNF-a and IL-1b. In contrast, even
though increased nuclear localization of NF-kB subunit p65 was
promoted by IL-17, this response was not robust upon stimulation
with IL-32 in both cell types (Fig. 5). Furthermore, both IL-32 and
IL-17 induced direct activation of NF-kB in a rabbit synovial fi-
broblast cell line (Fig. 5C). Taken together, these results indicated
that both IL-32 and IL-17 can activate the inflammatory tran-
scription factor NF-kB; however, this activity may involve dif-
ferent NF-kB subunit dimers.
Overall, similarities in downstream responses upon stimulation
with IL-32 and IL-17 in both macrophages and synoviocytes
supported the presence of common signaling intermediates for
Alteration of IL-32– and IL-17–induced responses upon
silencing of p300 and DAPK-1 genes
To establish the functional relevance of the identified common
targets, p300 and DAPK-1, we evaluated the impact of the
knockdown of these proteins on cellular responses induced by
IL-32 and IL-17, as well as those induced by proinflammatory
cytokines TNF-a and IL-1b. Accell SMARTpool EP300 (p300)
siRNA, DAPK-1 siRNA, and an NSC siRNA were used to knock
down the respective proteins in human macrophage-like THP-1
cells. Transcriptional analysis by qRT-PCR showed that gene ex-
pression for p300 and DAPK-1 was significantly (p , 0.01)
suppressed, by .60 6 5%, on treatment with the respective
siRNA and remained unaltered in cells treated with NSC, when
compared with control cells not treated with siRNA (Fig. 6A).
Immunoblots probing with specific Abs revealed a significant
knockdown of both p300 (EP300) and DAPK-1 proteins in cells
treated with the respective siRNAs, compared with cells treated
with NSC (Fig. 6B). Knockdown of protein expression was higher
than that observed at the mRNA level (Fig. 6A, 6B). Knockdown
of either p300 or DAPK-1 was not lethal to the cells, as deter-
mined by monitoring lactate dehydrogenase release for cellular
cytoxicity (data not shown).
We have shown that IL-32 can induce both IL-8 message (Fig.
4A) and protein production (Fig. 1B) in macrophage-like THP-1
cells. Even though IL-17 did not significantly enhance IL-8 gene
expression (Fig. 4A), IL-8 protein production was significantly
induced following stimulation with IL-17 (Fig. 1C) in
macrophage-like THP-1 cells. These results are consistent with
previous studies demonstrating that IL-17 alone is a poor stimulus
for inducing certain gene expressions, and that downstream pro-
duction of certain proinflammatory cytokines and chemokines,
including IL-8, is due to posttranscriptional effects exerted by IL-
17, primarily by stabilizing mRNA (25, 26). Taking these findings
together, we believed it valid to use IL-8 protein production as
a read-out for assessing the impact of p300 and DAPK-1 knock-
down on IL-32– and IL-17–induced downstream responses.
Knockdown of p300 suppressed chemokine IL-8 production
.80% in the presence of either IL-32 or IL-17 (Fig. 6C).
Knockdown of DAPK-1 abrogated the production of IL-8 upon
stimulation with IL-32 and significantly (p , 0.05) suppressed IL-
17–induced IL-8 production, by .70% (Fig. 6C). In addition,
knockdown of DAPK-1 significantly suppressed the production of
IL-8, by .90%, upon stimulation with proinflammatory cytokines
TNF-a and IL-1b (Fig. 6C). Similarly, knockdown of p300 sig-
nificantly (p , 0.05) suppressed IL-1b–induced production of
TNF-a, by .50% (Fig. 6D). In contrast, knockdown of either
32 and IL-17: Human macrophage-like THP-1 cells were stimulated with
either IL-32or IL-17(20ng/mleach) for15 min,followedby probingofthe
cell lysates in immunoblots with specific Abs to human phospho-p300
representative of three independent experiments. B, Densitometric analysis;
ratio of band density of cytokine-treatedsamples overunstimulated cells (y-
axis), calculated after normalization to band density of nonphosphorylated
forms of the respective proteins for each sample. Results represent an av-
erage of three independent experiments 6 SE. *p , 0.05.
Phosphorylation of p300 and DAPK-1 in the presence of IL-
Table II. Summary of primers used for qRT-PCR
GeneForward Primer (59–39)Reverse Primer (59–39)
The Journal of Immunology7131
p300 or DAPK-1 did not alter LPS-induced responses in
macrophage-like THP-1 cells (Fig. 6C, 6D).
from a dynamic and complex interplay of regulatory networks of
signaling pathways. The complexity of these interactions can limit
the efficacy of targeting a single molecule or one specific pathway
for the pharmacologic management of these disorders. For ex-
ample, in RA many patients do not respond to biologic therapies
that target the TNF-mediated pathway, and these therapeutics do
not completely control the progression of disease (5, 6). This
finding suggests that despite the central role proposed for TNF-a
in the pathogenesis of RA, there exists a complex network of
various cytokine-mediated regulatory pathways contributing to
the inflammatory microenvironment. Identification of overlapping
nodes within the different cytokine-mediated networks could be
valuable as potential drug targets for diseases characterized by
In this study, we investigated the molecular mechanisms in-
duced by two new cytokines, IL-32 and IL-17. These cytokines are
emerging as critical contributors to the pathogenesis of various
chronic inflammatory and autoimmune diseases. IL-32 is a newly
described potent proinflammatory cytokine, elevated levels of
which have been directly correlated to severity of chronic in-
flammatory disorders, including RA and IBD (9, 27). However, the
network of regulatory signaling pathways induced by IL-32 has
yet to be completely defined. In contrast, molecular regulation
mediated by IL-17 has been relatively well characterized (7, 28,
29). IL-17 is known to be a potent mediator of proinflamma-
tory cytokines, chemokines, acute phase response elements, and
defensins (7, 14, 30). IL-17 is associated with the pathophysiology
of autoimmune diseases, including RA, IBD, systemic lupus,
psoriasis, and autoimmune encephalitis (reviewed in Ref. 30).
Recently, this cytokine was also shown to be expressed in ath-
erosclerotic plaques (31). Despite similarities in proinflammatory
functions of IL-32 and IL-17, and both being described as thera-
peutic cytokine targets (14, 32), previous reports have speculated
that responses mediated by these cytokines may be differentially
dependent on the TNF pathway. The role of IL-32 in mediating
influx of inflammatory cells and subsequent cartilage damage in
macrophage-like THP-1 cells (B) were stimulated with either IL-32 (20 ng/
ml), IL-17 (20 ng/ml), TNF-a (10 ng/ml), or IL-1b (10 ng/ml) for 30 min.
Equivalent loading of nuclear extracts (5–8 mg) was probed in immuno-
blots with Abs specific to either NF-kB subunits p50, p65, or HDAC-1 as
input control. Immunoblot shown is representative of at least three in-
dependent experiments, for FLS independent experiments refer to cells
isolated from different independent donors. C, Cells from rabbit synovial
fibroblast cell line HIG-82 were transiently transfected with pNFkB-
MetLuc2-Reporter Vector (Clontech). The cells were stimulated with
cytokines—either TNF-a (10 ng/ml), IL-1b (10 ng/ml), IL-17 (20 ng/ml),
or IL-32 (20 ng/ml). The activation of NF-kB was monitored by employing
the Ready-To-Glow Secreted NF-kB Luciferase Reporter Assay (Clon-
tech), per the manufacturer’s instructions, after 4 and 6 h of stimulation.
Results represent luminescence over background levels in unstimulated
cells. Results are an average of at least five independent experiments 6 SE.
*p , 0.05, **p , 0.01.
Activation of transcription factor NF-kB. Human FLS (A) or
Human macrophage-like THP-1 cells were pretreated with mAb specific
for TNF-R1 (20 mg/ml each) for 1 h, followed by stimulation with cyto-
kines—either IL-32 (20 ng/ml), IL-17 (20 ng/ml), TNF-a (10 ng/ml), or
IL-1b (10 ng/ml). Transcriptional responses were evaluated for chemokine
Gro-a (A) and proinflammatory cytokine IL-23 (B) after 4 h of stimulation
by qRT-PCR. Fold changes (y-axis) for each gene was normalized to 18S
RNA and is represented relative to gene expression in unstimulated cells
normalized to 1, using the comparative Ct method. TC supernatants were
monitored after 24 h of stimulation for the production of chemokines Gro-
a (C) and IL-8 (D) by ELISA. Protein production monitored in the TC
supernatants by ELISA is shown after subtraction of background levels
found in unstimulated control cells. Results represent an average of three
independent experiments 6 SE. *p , 0.05, **p , 0.01.
Alteration of responses in the presence of TNF-R1 mAb.
32. Human macrophage-like THP-1 cells (in vitro) (A) and human FLS
isolated from synovial tissues (ex vivo) (B) were stimulated with either IL-
17 or IL-32 (20 ng/ml each) for 4 h. RNAwas isolated, and transcriptional
responses were analyzed by qRT-PCR. Fold changes (y-axis) for each gene
were calculated after normalization to housekeeping gene 18S RNA and
quantitated relative to gene expression in unstimulated cells normalized to
1, using the comparative Ct method. Results represent an average of four
independent experiments (FLS isolated from four independent donors) 6
SE. *p , 0.05, **p , 0.01, ***p , 0.001.
Transcriptional responses in the presence of IL-17 and IL-
7132 COMMON SIGNALING INTERMEDIATES OF IL-32 AND IL-17
arthritis is dependent on TNF-related mechanisms (17); in con-
trast, IL-17–mediated inflammation under arthritic conditions is,
in part, TNF independent (13). To date, no studies have been done
that provide insight into the differential regulatory processes or
explain the basis for the differential TNF pathway dependence
in IL-32– and IL-17–mediated inflammatory responses. The key
findings in this study were 1) IL-32–mediated responses, but not
IL-17–mediated ones, may be dependent on TNF-R1 (Fig. 3,
Table I); and 2) p300 and DAPK-1 are common signaling inter-
mediates for both IL-32 and IL-17 (Fig. 2, Table I), and knock-
down of these proteins impaired the cytokine-mediated down-
stream responses (Fig. 6). These results suggest that, even though
IL-32 and IL-17 may have differential dependence on the TNF-
pathway, their induced inflammatory networks overlap, and that
there are common downstream targets of these cytokines.
We have shown that, unlike IL-17–mediated responses, cytokine
IL-32–mediated downstream cellular responses were dependent
on TNF-R1 (Fig. 3), which supported the kinome analysis (Table
I). However, different amounts of TNF-a may be produced fol-
lowing stimulation with 20 ng/ml of IL-17 and IL-32 in
macrophage-like THP-1 cells (as indicated by the results shown in
Fig. 4A), which could also have an impact on the differential
effects seen with TNF-R1 blocking in the presence of these
cytokines. TNF-a stimulation was used as a positive control for
the TNF-R1 neutralization assays. As expected, blocking of TNF-
R1 suppressed TNF-a–induced transcriptional responses and Gro-
a production but did not inhibit IL-8 production (Fig. 3D). Two
receptors with distinct signaling mechanisms have been defined
for the proinflammatory events induced in the presence of the
cytokine TNF-a, TNF-R1 (TNFRSF1A or p55), and TNF-R2
(TNFRSF1B or p75). TNF-a–induced IL-8 production upon
blocking TNF-R1 indicates that this response is probably medi-
ated by the TNF-R2 signaling pathway. These results also suggest
that IL-32–induced responses are not a result of the TNF-a
feedback loop and may be directly dependent on TNF-R1 activity.
Therefore, it may be speculated that the TNF pathway-dependent
role of IL-32 in chronic inflammation is due to the engagement of
the cytokine with TNF-R1, which warrants further investigation.
The only direct interacting protein partner demonstrated for IL-32
is a neutrophil-derived serine protease, proteinase 3 (33). To date,
no other receptor has been described for IL-32. This study sug-
gests that IL-32–mediated responses are dependent on TNF-R1,
and thus provides molecular insight for the speculation that in-
flammatory functions of IL-32 are, in part, dependent on the TNF
We identified p300 and DAPK-1 as common protein phos-
phorylation targets for IL-32 and IL-17. Our results indicate that
there may be overlap in the inflammatory networks induced by
these two cytokines, as well as commonalities in the regulatory
processes triggered by these cytokines. The functional relevance
of the identified common protein targets was confirmed using
knockdown studies, which showed that knockdown of either p300
either human p300, human DAPK-1, or NSC in Accell delivery media for 96 h. Knockdown efficiency was evaluated by qRT-PCR for mRNA expression
(A) and immunoblots (B) by probing the nuclear extracts with specific Ab to human p300, and total cell extracts with Ab to human DAPK-1. Abs specific to
human HDAC and GAPDH were used to estimate loading controls, respectively. The knockdown cells were stimulated with either IL-32 (20 ng/ml), IL-17
(20 ng/ml), IL-1b (10 ng/ml), TNF-a (10 ng/ml), or LPS (10 ng/ml) for 24 h. TC supernatants were monitored for the production of chemokine IL-8 (C)
and cytokine TNF-a (D) by ELISA. Results are representative of at least three independent experiments 6 SE. *p , 0.05, **p , 0.01.
Knockdown of p300 and DAPK-1 in macrophages. Human macrophage-like THP-1 cells were treated with Accell SMARTpool siRNA for
The Journal of Immunology 7133
or DAPK-1 altered both IL-32– and IL-17–induced downstream
responses (Fig. 6C). In addition, knockdown of p300 and DAPK-1
also altered certain TNF- and IL-1b–induced cellular responses
(Fig. 6C, 6D). These results were consistent with our hypothesis
that common protein targets of IL-32 and IL-17 would likely be
involved in TNF-dependent and -independent cellular responses.
The transcriptional coactivator p300, in concert with other
transcription factors, triggers gene expression in the presence of
inflammatory stimuli. For example, p300 is essential for IL-1b–
induced PG release and for phospholipase A(2) and NF-kB acti-
vation in human tracheal smooth muscles (34). We showed that
IL-32 and IL-17 stimulation resulted in the phosphorylation of
p300 at Ser1834(Fig. 2, Table I). A previous report has demon-
strated that phosphorylation of p300 at Ser1834results from the
nuclear translocation of AKT upon the activation of the PI3K/
AKT pathway in response to proinflammatory cytokine TNF-a
(35). Similarly, the involvement of activation of the AKT pathway
upon stimulation with IL-32 has been recently demonstrated in
osteoclasts (12), as it has for IL-17 in retinal astrocytes and
bronchial epithelial cells (28, 36). With the data taken together, we
can therefore speculate that the cytokines IL-32 and IL-17 induce
the phosphorylation of p300 at Ser1834by engaging the PI3K/AKT
pathway, resulting in the activation of NF-kB.
The second protein target identified for cytokines IL-32 and IL-
17 in this study was DAPK-1, which is an apoptotic regulator and
has been implicated in the disease process of several cancers (37–
39). IL-32 is known to promote apoptosis of keratinocytes in
atopic dermatitis (11), which supports our results that identified an
apoptosis regulator, DAPK-1, as a protein target for IL-32. An-
other study has shown that inflammation can lead to aberrant DNA
methylation of the DAPK-1 gene in a model of IBD (40). In ad-
dition, DAPK has been recently described as a potential thera-
peutic target for chronic neurodegenerative diseases (41). Because
dysregulation of inflammation has been correlated with several
cancers, as well as with neurodegenerative disorders such as
Alzheimer’s disease, it may be speculated that DAPK-1 plays
a regulatory role in pathways induced in the presence of chronic
inflammatory stimuli, such as the cytokines investigated in this
In summary, this study provided molecular insight into the TNF
pathway-dependent role of the IL-32–mediated inflammatory
network, demonstrating that IL-32–induced cellular responses
could be dependent on TNF-R1, which contrasts with the activity
of IL-17. Despite differential dependence on TNF-R1 activity, we
suggest that the common signaling intermediates p300 and DAPK-
1 represent nodes where the inflammatory networks mediated by
IL-32 and IL-17 overlap, leading to the convergence of signaling
in the activation of NF-kB and resulting in common downstream
proinflammatory cellular responses. We can also speculate that
p300 and DAPK-1 may be pivotal checkpoints for TNF-dependent
and -independent inflammatory networks. This discovery merits
further investigation to elucidate the role of p300 and DAPK-1
as potential therapeutic targets for immune-mediated chronic in-
flammatory diseases, especially for nonresponders of anti-TNF
We thank Keng Wong for technical support and Drs. John Wilkins and Dus-
tin Lippert (Manitoba Centre for Proteomics and Systems Biology, Univer-
sity of Manitoba) for intellectual input and critical discussions.
The authors have no financial conflicts of interest.
1. Feldmann, M., F. M. Brennan, and R. N. Maini. 1996. Role of cytokines in
rheumatoid arthritis. Annu. Rev. Immunol. 14: 397–440.
2. Martel-Pelletier, J., D. J. Welsch, and J. P. Pelletier. 2001. Metalloproteases and
inhibitors in arthritic diseases. Best Pract. Res. Clin. Rheumatol. 15: 805–829.
3. Padyukov, L., J. Lampa, M. Heimbu ¨rger, S. Ernestam, T. Cederholm,
I. Lundkvist, P. Andersson, Y. Hermansson, A. Harju, L. Klareskog, and J. Bratt.
2003. Genetic markers for the efficacy of tumour necrosis factor blocking
therapy in rheumatoid arthritis. Ann. Rheum. Dis. 62: 526–529.
4. van den Berg, W. B. 2000. Arguments for interleukin 1 as a target in chronic
arthritis. Ann. Rheum. Dis. 59(Suppl 1): i81–i84.
5. Huber, L. C., O. Distler, I. Tarner, R. E. Gay, S. Gay, and T. Pap. 2006. Synovial
fibroblasts: key players in rheumatoid arthritis. Rheumatology (Oxford) 45: 669–
6. Choy, E. H., and G. S. Panayi. 2001. Cytokine pathways and joint inflammation
in rheumatoid arthritis. N. Engl. J. Med. 344: 907–916.
7. Gaffen, S. L. 2004. Biology of recently discovered cytokines: interleukin-17—
a unique inflammatory cytokine with roles in bone biology and arthritis. Arthritis
Res. Ther. 6: 240–247.
8. Dinarello, C. A., and S. H. Kim. 2006. IL-32, a novel cytokine with a possible
role in disease. Ann. Rheum. Dis. 65(Suppl 3): iii61–iii64.
9. Joosten, L. A., M. G. Netea, S. H. Kim, D. Y. Yoon, B. Oppers-Walgreen,
T. R. Radstake, P. Barrera, F. A. van de Loo, C. A. Dinarello, and W. B. van den
Berg. 2006. IL-32, a proinflammatory cytokine in rheumatoid arthritis. Proc.
Natl. Acad. Sci. USA 103: 3298–3303.
10. Calabrese, F., S. Baraldo, E. Bazzan, F. Lunardi, F. Rea, P. Maestrelli, G. Turato,
K. Lokar-Oliani, A. Papi, R. Zuin, et al. 2008. IL-32, a novel proinflammatory
cytokine in chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care
Med. 178: 894–901.
11. Meyer, N., M. Zimmermann, S. Burgler, C. Bassin, S. Woehrl, K. Moritz,
C. Rhyner, P. Indermitte, P. Schmid-Grendelmeier, M. Akdis, et al. 2010. IL-32
is expressed by human primary keratinocytes and modulates keratinocyte apo-
ptosis in atopic dermatitis. J. Allergy Clin. Immunol. 125: 858–865.e810.
12. Mabilleau, G., and A. Sabokbar. 2009. Interleukin-32 promotes osteoclast dif-
ferentiation but not osteoclast activation. PLoS ONE 4: e4173.
13. Koenders, M. I., E. Lubberts, F. A. van de Loo, B. Oppers-Walgreen, L. van den
Bersselaar, M. M. Helsen, J. K. Kolls, F. E. Di Padova, L. A. Joosten, and
W. B. van den Berg. 2006. Interleukin-17 acts independently of TNF-alpha under
arthritic conditions. J. Immunol. 176: 6262–6269.
14. Lubberts, E. 2008. IL-17/Th17 targeting: on the road to prevent chronic de-
structive arthritis? Cytokine 41: 84–91.
15. Korn, T., E. Bettelli, M. Oukka, and V. K. Kuchroo. 2009. IL-17 and Th17 cells.
Annu. Rev. Immunol. 27: 485–517.
16. Koenders, M. I., L. A. Joosten, and W. B. van den Berg. 2006. Potential new
targets in arthritis therapy: interleukin (IL)-17 and its relation to tumour necrosis
factor and IL-1 in experimental arthritis. Ann. Rheum. Dis. 65(Suppl 3): iii29–
17. Shoda, H., K. Fujio, Y. Yamaguchi, A. Okamoto, T. Sawada, Y. Kochi, and
K. Yamamoto. 2006. Interactions between IL-32 and tumor necrosis factor alpha
contribute to the exacerbation of immune-inflammatory diseases. Arthritis Res.
Ther. 8: R166.
18. Mookherjee, N., K. L. Brown, D. M. Bowdish, S. Doria, R. Falsafi, K. Hokamp,
F. M. Roche, R. Mu, G. H. Doho, J. Pistolic, et al. 2006. Modulation of the TLR-
mediated inflammatory response by the endogenous human host defense peptide
LL-37. J. Immunol. 176: 2455–2464.
19. Jalal, S., R. Arsenault, A. A. Potter, L. A. Babiuk, P. J. Griebel, and S. Napper.
2009. Genome to kinome: species-specific peptide arrays for kinome analysis.
Sci. Signal. 2: pl1.
20. Turner, N. A., R. S. Mughal, P. Warburton, D. J. O’Regan, S. G. Ball, and
K. E. Porter. 2007. Mechanism of TNFalpha-induced IL-1alpha, IL-1beta and
IL-6 expression in human cardiac fibroblasts: effects of statins and thiazolidi-
nediones. Cardiovasc. Res. 76: 81–90.
21. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-
time RT-PCR. Nucleic Acids Res. 29: e45.
22. Kim, S. H., S. Y. Han, T. Azam, D. Y. Yoon, and C. A. Dinarello. 2005.
Interleukin-32: a cytokine and inducer of TNFalpha. Immunity 22: 131–142.
23. Cohen, P. 2002. Protein kinases—the major drug targets of the twenty-first
century? Nat. Rev. Drug Discov. 1: 309–315.
24. Sawyers, C. L. 2002. Rational therapeutic intervention in cancer: kinases as drug
targets. Curr. Opin. Genet. Dev. 12: 111–115.
25. Henness, S., E. van Thoor, Q. Ge, C. L. Armour, J. M. Hughes, and A. J. Ammit.
2006. IL-17A acts via p38 MAPK to increase stability of TNF-alpha-induced IL-
8 mRNA in human ASM. Am. J. Physiol. Lung Cell. Mol. Physiol. 290: L1283–
26. Hartupee, J., C. Liu, M. Novotny, X. Li, and T. Hamilton. 2007. IL-17 enhances
chemokine gene expression through mRNA stabilization. J. Immunol. 179:
27. Shioya, M., A. Nishida, Y. Yagi, A. Ogawa, T. Tsujikawa, S. Kim-Mitsuyama,
A. Takayanagi, N. Shimizu, Y. Fujiyama, and A. Andoh. 2007. Epithelial
overexpression of interleukin-32alpha in inflammatory bowel disease. Clin. Exp.
Immunol. 149: 480–486.
28. Huang, F., C. Y. Kao, S. Wachi, P. Thai, J. Ryu, and R. Wu. 2007. Requirement
for both JAK-mediated PI3K signaling and ACT1/TRAF6/TAK1-dependent NF-
kappaB activation by IL-17A in enhancing cytokine expression in human airway
epithelial cells. J. Immunol. 179: 6504–6513.
29. Iwanami, K., I. Matsumoto, Y. Tanaka-Watanabe, A. Inoue, M. Mihara,
Y. Ohsugi, M. Mamura, D. Goto, S. Ito, A. Tsutsumi, et al. 2008. Crucial role of
7134COMMON SIGNALING INTERMEDIATES OF IL-32 AND IL-17
the interleukin-6/interleukin-17 cytokine axis in the induction of arthritis by
glucose-6-phosphate isomerase. Arthritis Rheum. 58: 754–763.
30. Onishi, R. M., and S. L. Gaffen. 2010. Interleukin-17 and its target genes:
mechanisms of interleukin-17 function in disease. Immunology 129: 311–321.
31. de Boer, O. J., J. J. van der Meer, P. Teeling, C. M. van der Loos, M. M. Idu,
F. van Maldegem, J. Aten, and A. C. van der Wal. 2010. Differential expression
of interleukin-17 family cytokines in intact and complicated human athero-
sclerotic plaques. J. Pathol. 220: 499–508.
32. Asquith, D. L., and I. B. McInnes. 2007. Emerging cytokine targets in rheu-
matoid arthritis. Curr. Opin. Rheumatol. 19: 246–251.
33. Novick, D., M. Rubinstein, T. Azam, A. Rabinkov, C. A. Dinarello, and
S. H. Kim. 2006. Proteinase 3 is an IL-32 binding protein. Proc. Natl. Acad. Sci.
USA 103: 3316–3321.
34. Lee, C. W., I. T. Lee, C. C. Lin, H. C. Lee, W. N. Lin, and C. M. Yang. 2010.
Activation and induction of cytosolic phospholipase A2 by IL-1beta in human
tracheal smooth muscle cells: role of MAPKs/p300 and NF-kappaB. J. Cell.
Biochem. 109: 1045–1056.
35. Huang, W. C., and C. C. Chen. 2005. Akt phosphorylation of p300 at Ser-1834 is
essential for its histone acetyltransferase and transcriptional activity. Mol. Cell.
Biol. 25: 6592–6602.
36. Ke, Y., G. Jiang, D. Sun, H. J. Kaplan, and H. Shao. 2009. Retinal astrocytes
respond to IL-17 differently than retinal pigment epithelial cells. J. Leukoc. Biol.
37. Kim, D. H., H. H. Nelson, J. K. Wiencke, D. C. Christiani, J. C. Wain, E. J. Mark,
and K. T. Kelsey. 2001. Promoter methylation of DAP-kinase: association with
advanced stage in non-small cell lung cancer. Oncogene 20: 1765–1770.
38. Kuester, D., A. A. Dar, C. C. Moskaluk, S. Krueger, F. Meyer, R. Hartig,
M. Stolte, P. Malfertheiner, H. Lippert, A. Roessner, et al. 2007. Early in-
volvement of death-associated protein kinase promoter hypermethylation in the
carcinogenesis of Barrett’s esophageal adenocarcinoma and its association with
clinical progression. Neoplasia 9: 236–245.
39. Raval, A., S. M. Tanner, J. C. Byrd, E. B. Angerman, J. D. Perko, S. S. Chen,
B. Hackanson, M. R. Grever, D. M. Lucas, J. J. Matkovic, et al. 2007. Down-
regulation of death-associated protein kinase 1 (DAPK1) in chronic lymphocytic
leukemia. Cell 129: 879–890.
40. Hahn, M. A., T. Hahn, D. H. Lee, R. S. Esworthy, B. W. Kim, A. D. Riggs,
F. F. Chu, and G. P. Pfeifer. 2008. Methylation of polycomb target genes in
intestinal cancer is mediated by inflammation. Cancer Res. 68: 10280–10289.
41. Cuny, G. D. 2009. Kinase inhibitors as potential therapeutics for acute and
chronic neurodegenerative conditions. Curr. Pharm. Des. 15: 3919–3939.
The Journal of Immunology7135