il-33/st2 axis in inflammation and immunopathology
TRANSCRIPT
IMMUNOLOGY IN SERBIA
IL-33/ST2 axis in inflammationand immunopathology
Marija Milovanovic • Vladislav Volarevic •
Gordana Radosavljevic • Ivan Jovanovic •
Nada Pejnovic • Nebojsa Arsenijevic • Miodrag L. Lukic
Published online: 6 March 2012
� Springer Science+Business Media, LLC 2012
Abstract Interleukin-33 (IL-33), a member of the IL-1 family of cytokines, binds to its plasma membrane receptor,
heterodimeric complex consisted of membrane-bound ST2L and IL-1R accessory protein, inducing NFkB and MAPK
activation. IL-33 exists as a nuclear precursor and may act as an alarmin, when it is released after cell damage or as
negative regulator of NFjB gene transcription, when acts in an intracrine manner. ST2L is expressed on several immune
cells: Th2 lymphocytes, NK, NKT and mast cells and on cells of myeloid lineage: monocytes, dendritic cells and
granulocytes. IL-33/ST2 axis can promote both Th1 and Th2 immune responses depending on the type of activated cell and
microenvironment and cytokine network in damaged tissue. We previously described and discuss here the important role of
IL-33/ST2 axis in experimental models of type 1 diabetes, experimental autoimmune encephalomyelitis, fulminant hep-
atitis and breast cancer. We found that ST2 deletion enhance the development of T cell-mediated autoimmune disorders,
EAE and diabetes mellitus type I. Disease development was accompanied by dominantly Th1/Th17 immune response but
also higher IL-33 production, which suggest that IL-33 in receptor independent manner could promote the development of
inflammatory autoreactive T cells. IL-33/ST2 axis has protective role in Con A hepatitis. ST2-deficient mice had more
severe hepatitis with higher influx of inflammatory cells in liver and dominant Th1/Th17 systemic response. Pretreatment
of mice with IL-33 prevented Con A-induced liver damage through prevention of apoptosis of hepatocytes and Th2
amplification. Deletion of IL-33/ST2 axis enhances cytotoxicity of NK cells, production of IFN-c in these cells and
systemic production of IFN-c, IL-17 and TNF-a, which leads to attenuated tumor growth. IL-33 treatment of tumor-bearing
mice suppresses activity of NK cells, dendritic cell maturation and enhances alternative activation of macrophages. In
conclusion, we observed that IL-33 has attenuated anti-inflammatory effects in T cell-mediated responses and that both
IL-33 and ST2 could be further explored as potential therapeutic targets in treatment of immune-mediated diseases.
Keywords IL-33/ST2 axis � Con A hepatitis � EAE � MLD-STZ diabetes � Mouse breast cancer
IL-33/ST2 axis
Interleukin-33 (IL-33) is a member of the IL-1 cytokine
family, originally described as a nuclear protein in cerebral
arteries [1] and later as NF-HEV, a nuclear factor
expressed in human high endothelial venules in secondary
lymphoid organs [2]. Recently, IL-33 was identified as the
ligand for the orphan receptor, ST2 (IL-1RL1). ST2 mol-
ecule is a member of the IL-1 receptor family [3] that exists
in two forms: a transmembrane full-length form (ST2L)
and a soluble, secreted form (sST2) due to differential
splicing of ST2 mRNA [4]. Soluble ST2 acts as a decoy
receptor for IL-33 [5]. In normal conditions, the serum
concentration of soluble ST2 is below the detectable level,
but elevated level of ST2 has been reported in patients with
autoimmune diseases [6], asthma [7], idiopathic pulmonary
fibrosis [8], myocardial infarction and heart failure [9].
M. Milovanovic � V. Volarevic � G. Radosavljevic �I. Jovanovic � N. Pejnovic � N. Arsenijevic � M. L. Lukic (&)
Faculty of Medicine, Center for Molecular Medicine and Stem
Cell Research, University of Kragujevac, Svetozara Markovica
69, 34000 Kragujevac, Serbia
e-mail: [email protected]
Miodrag L. Lukic
123
Immunol Res (2012) 52:89–99
DOI 10.1007/s12026-012-8283-9
ST2L is expressed by many hematopoietic cells, NK and
NKT cells, mast cells, monocytes, dendritic cells and
granulocytes and selectively expressed by murine and
human Th2 cells but not by Th1 lymphocytes [10]. ST2
associates with IL-1R accessory protein (IL-1RAcP) to
form an IL-33 receptor (IL-33R1) [11], and IL-33 signals
via this heterodimer. Soluble form of IL-1RAcP interacts
with the sST2–IL-33 complex to increase blocking of
IL-33 signaling [12]. There is another IL-1R family mem-
ber, SIGIRR, which, accompanied by ST2L (IL-33R2),
negatively regulates IL-33 effects [13].
The binding of IL-33 to IL-33 receptor results in the
recruitment of myeloid differentiation primary response
protein 88 (MyD88), IL-1R-associated kinase 1 (IRAK1)
and IRAK4 to the receptor complex in cytoplasmic region
of ST2, which induces activation of various signaling
proteins, including nuclear factor-jB (NF-jB), inhibitor of
NF-jB-a (IjBa) and extracellular signal-regulated kinase 1
(eRK1), eRK2, p38 and c-Jun N-terminal kinase (JNK)
leading to the induction of inflammatory mediators IL-1b,
IL-3, IL-6, TNF, IL-5 and IL-13 [14–16].
At the mRNA level, IL-33 is expressed in many organs
[11] in humans and mice. However, at the protein level, IL-
33 is mainly and constitutively expressed in epithelial and
endothelial cells [17]. Immune cells, macrophages and
dendritic cells also produce IL-33 after adequate stimulation
[18]. Pathogen-associated molecular pattern (PAMP) mol-
ecules and also cytokines TNF-a, IL-1 and IFN-c stimulate
the production of IL-33 in macrophages [19, 20]. On the
other hand, some proinflammatory cytokines such as TNF-aand IL-6 are also potent inducers of soluble ST2 (sST2) [21],
which block the effects of IL-33. Analogous to other IL-1
family cytokines, it was proposed that IL-33 becomes acti-
vated by caspase activity, but pro-IL-33 does not have a
typical cleavage site seen in pro-IL-1b and IL-18. Later
findings indicated that caspase-1, caspase-3 and caspase-7
[22–24] released after apoptotic cell death inactivated IL-33
and that IL-33 is active in pro-form. IL-33 has no leader
sequence and it is not clear, at present, how it is released
from cells [25]. It was shown that biologically active pro-IL-
33 can be released by necrotic cells as ‘‘alarmin’’ [26]. Since
IL-33 is mainly expressed in lining, epithelial and endo-
thelial cells [17] and is released after cell damage, it is
proposed to have an important role in sensing damage in
various infectious and inflammatory diseases. In the absence
of proinflammatory stimuli, IL-33 is localized to the nucleus
[2, 27]. Additionally, IL-33, as a full-length protein, can act
in an intracrine manner, translocating to the nucleus, where
it binds to the chromatin and stimulates expression of IkBa,
TNF-a, and C-REL [27, 28]. Since it has been shown that IL-
33 is released from necrotic cells of structural but not
hematopoietic origin [29], intracrine action of IL-33 could
be the main way of action in immune cells.
However, it is well known that IL-33 affects the func-
tion of cells that express ST2 molecule. IL-33 polarizes
naive T cells to produce Th2-associated cytokines IL-4,
IL-5 and IL-13 [11] and functions as a chemoattractant for
Th2 cells in vitro and in vivo [30], but also induces
secretion of proinflammatory cytokines and chemokines by
mast cells [16], basophils [31] and Th1 type cytokines from
NK and NKT cells [31, 32]. Also, IL-33 amplifies polari-
zation of alternatively activated M2 macrophages [33],
induces maturation of dendritic cells [34] and may promote
Th1-type response depending on the local cytokine milieu
[35].
IL-33/ST2 axis as the first line of defense in infection
IL-33, as a full-length molecule, is mainly expressed in
epithelial and endothelial cells [17, 36] especially in high
endothelial venules [27], and it is proposed that IL-33
serves as the first line of defense against microbes that
shape adaptive immune response. Humphreys et al. [37]
were the first to show that IL-33 protects from parasitic
infection. They demonstrated increased expression of IL-
33 mRNA in the colon of resistant, but not susceptible
mice after infection with Trichiuris muris. Exogenous IL-
33 administered to susceptible mice made them resistant to
T. muris infection. IL-33 acts as the initial innate signal of
parasite invasion to the host that polarizes adaptive
immune response toward Th2-biased response, which is
host protective in this disease. It also appears that IL-33/
ST2 axis affects significantly innate as well as adaptive
immune response, as IL-33 was unable to induce parasite
expulsion in SCID mice. However, IL-33 can exacerbate
the pathology associated with chronic T. muris infection
through T cell-independent mechanisms, probably
increasing the production of IFN-c in NK cells [2]. Regu-
latory role of IL-33 in infection is also shown during
Toxoplasma gondii infection [38]. ST2 mRNA expression
was upregulated, but IL-33 mRNA expression was not
altered, in brain lesions of mice infected with T. gondii
compared with naive mice [38]. However, ST2 knockout
mice had infection with T. gondii with more severe
pathology and increased mRNA levels of iNOS, IFN-c and
TNF-a in the central nervous system [37].
IL-33/ST2 signaling affects immune response to viruses.
The serum levels of s ST2 protein were increased in
patients infected with dengue virus [39]. Infection of mice
with Theiler’s murine encephalomyelitis virus is followed
by increased expression of IL-33 in the glia, which is fol-
lowed by enhanced innate effector functions of glial cells,
which suggested that IL-33 has important role in host
defense in the CNS [38]. The role of IL-33 in antiviral
defense was also shown in respiratory syncytial virus
90 Immunology in Serbia (2012) 52:89–99
123
infection in mice. Treatment with monoclonal ST2-specific
antibody reduced lung inflammation and disease severity in
mice with Th2 type of immune response and clinical signs
of bronchiolitis [40].
IL-33/ST2 axis in fulminant hepatitis
Recently, it has been demonstrated that both IL-33 and
soluble ST2 were elevated in sera of patients with liver
failure [41]. Therefore, we decided to dissect out the role of
IL-33/ST2 axis in liver pathology by using experimental
model of acute liver injury, concanavalin A (Con A)-
induced hepatitis. Con A-induced liver injury is well-
established murine model of T cell-mediated hepatitis
[42–45]. Intravenous injection of Con A induces massive
necrosis of hepatocytes and immune cell activation that
resembles the pathology of immune-mediated fulminant
hepatitis in humans [46].
We demonstrated that IL-33/ST2 axis has protective role
in Con A hepatitis [47] showing that ST2 knockout mice
had more sever Con A-induced hepatitis than wild-type
(WT) animals. Liver damage in ST2 knockout mice was
accompanied by intrahepatic accumulation of macro-
phages, CD4? and CD8? T lymphocytes, NK and NKT
cells, while the number of CD4?Foxp3? regulatory T cells,
that have protective role in Con A-induced hepatitis [48,
49], was reduced in ST2-deficient mice. Further, ST2-/-
mice had altered systemic immune response toward Th1/
Th17 type: we found elevated levels of systemic proin-
flammatory cytokines (TNF alpha, IFN gamma and IL-17)
and attenuated serum level of IL-4 in ST2 knockout mice.
Elevated serum level of TNF-a correlated with higher
number of TNF-a producing CD4? T cells in livers of
ST2-/- mice, probably as a consequence of increased
capacity of CD4? T cells to secrete TNF in the absence of
T1/ST2 as was reported in mice treated with anti-T1/ST2
monoclonal antibodies [50]. It is well known that CD8?
effector T cells are potent producers of IFN-c [51], and that
IFN-c-producing NK and NKT cells are major effector
cells involved in Con A-induced liver injury [52, 53].
Consistent with these findings, we showed massive infil-
tration of IFN-c-producing CD8?T, NK and NKT cells in
livers of Con A-treated ST2-/- mice. Thus, we concluded
that IFN-c, absolutely necessary in the pathogenesis of Con
A hepatitis [54–56], was mainly produced by CD8?T
lymphocytes, NK and NKT cells. In Con A hepatitis, IL-17
has been reported to be both proinflammatory and without
a direct inflammation modulating role [57, 58]. In our
study, elevated serum levels of IL-17 correlated with
severe liver damage and massive infiltration of IL-17-
producing NKT cells in the liver. Recently published data
[59] confirm that IL-17 plays a pathological role in acute
liver damage. Xu et al. [59] demonstrated that a patho-
logical role of exogenous IL-23 in Con A hepatitis depends
on the production of IL-17, suggesting that in fulminate
hepatitis IL-17 plays a pathological role.
In line with these observations, we showed that pre-
treatment of WT mice with IL-33 attenuates Con
A-induced liver injury. The injection of single dose of IL-
33 significantly reduced Con A-induced liver damage in
wild-type BALB/c mice, prevented the recruitment of
mononuclear cells into the liver and increased the influx of
T regulatory cells. Further, levels of Th1 and Th17 type
cytokines in the serum of IL-33 pretreated mice were
decreased after Con A injection. Therefore, we assume that
the main mechanism by which IL-33/ST2 pathway has a
protective role in Con A-mediated liver injury is prevention
of Th1/Th17 cell-mediated hepatic immune response.
It is well known that after Con A injection, polyclonally
activated lymphocytes, particularly CD4? T lymphocytes
and NKT cells, stick to liver sinusoidal endothelium and
destroy SECs and underlying hepatocytes [46]. IL-33 is
proposed to be released as an alarmin from necrotic cells
while caspase-1, caspase-3 and caspase-7 released after
apoptotic cell death inactivate it [22–25]. Therefore, we
decided to investigate the influence of IL-33 on the
expression of pro- and anti-apoptotic genes during Con
A-induced liver injury. Pretreatment of WT BALB/c mice
with IL-33 suppressed the activation of pro-apoptotic cas-
pase-3 and mitochondrial BAX and enhanced the expres-
sion of anti-apoptotic Bcl-2 and p-ERK leading to the
attenuation of hepatitis. We and Erhardt and Tiegs [47, 60]
suggest that during acute hepatitis IL-33 is released from
damaged LSECs and hepatocytes, activates cells that
express ST2 molecule, particularly CD4? Th2 lympho-
cytes, NKT cells and activated macrophages, shifts
immune response toward Th2 type, suppresses caspase-3
activity and enhances expression of anti-apoptotic Bcl-2
and p-ERK that limits liver damage and promotes healing
of the liver tissue (illustrated in Fig. 1].
IL-33/ST2 axis in allergy and asthma
Asthma is a chronic inflammatory disease classically
characterized by increased Th2 cytokine production. IL-33
is a strong inducer of Th2 immune responses and its role in
immunopathology of asthma had been recently reviewed
elsewhere [61, 62]. Higher expression of IL-33 and sST2
was found in sera and endobronchial biopsies from asth-
matic patients [63] as well as in mouse models of asthma
induced by ovalbumin [64, 65] compared to healthy con-
trols. In the lung tissue, IL-33 and ST2 were mainly
expressed in bronchial epithelial cells [66]. Nevertheless,
the precise role of IL-33 and ST2 in mouse models of
Immunology in Serbia (2012) 52:89–99 91
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asthma is unknown. There are the opposite results found in
the studies using ST2-deficient mice, mice treated with
anti-ST2 proteins or IL-33-deficient mice. Massive cell
influx in the lung and airway hyper-responsiveness in a
murine model of ovalbumin-induced airway inflammation
was found after intranasal administration of IL-33 [67, 68]
and in IL-33 transgenic mice [69]. IL-33 potentiates mat-
uration of dendritic cells upregulating the expression of
CD80, CD40 and OX40L, accompanied by the release of
proinflammatory cytokines, IL-6, IL-1b, TNF-a, and IL-33
also induces allergen-specific proliferation of naive T cells.
IL-33 also affects migration of dendritic cells to the lymph
nodes, where they can contribute to the priming of Th2
cells and the induction of allergic airway inflammation
[70]. In line with these findings, IL-33 knockout mice
sensitized with ovalbumin emulsified in alum showed
attenuated recruitment of inflammatory cells to the lung
and attenuated airway hyper-responsiveness [71]. Further-
more, application of blocking anti-ST2 antibodies or ST2-
Ig fusion protein inhibited eosinophilic pulmonary
inflammation and airways hyper-responsiveness [72]. In
contrast to these reports, ST2-deficient mice were not
protected in the ovalbumin-induced airway inflammation
model [73] but have attenuated inflammation in different
model of asthma. Further, an exacerbated disease was
found in wild-type or Rag-1-/- mice that had undergone
adoptive transfer of ST2-/- ovalbumin-specific Th2 cells
[74].
The reason for these differences is not clear. It could be
due to different expression of IL1RAcP in ST2-deficient
mice. IL1RAcP forms receptor complex not only with ST2
but also with IL-1 receptor (IL1R) and amplifies the signal
[75]. Thus, if there are no ST2 molecules on cell mem-
branes, IL-1 signaling could be overamplificated, inducing
inflammation. Or, if there is no soluble ST2 that blocks IL-
33, IL-33 can bind to some other receptor and induce
inflammation.
Anaphylaxis is characterized by elevated immunoglob-
ulin-E (IgE) antibodies that signal via the high affinity Fcereceptor (FceRI) to release inflammatory mediators [14].
IL-33 is markedly elevated in the serum of patients during
an anaphylactic shock and in inflamed skin tissue of
patients with atopic dermatitis. In the presence of IgE, IL-
33 activates mast cells and directly induces degranulation
following IgE sensitization. In animals that are systemi-
cally sensitized with IgE, IL-33 administration exacerbates
antigen-induced anaphylaxis and induces the degranulation
of IgE-sensitized mast cells in the skin even in the absence
of antigen [14].
IL-33/ST2 axis in T cell-mediated autoimmunity
We explored the effects of IL-33/ST2 signaling in several
autoimmune disorders mediated by T lymphocytes. Our
findings indicate that ST2 deletion and exclusion of IL-33/
Fig. 1 Proposed role of IL-33 in tissue regeneration in Con
A-induced hepatitis. Upon Con A injection, resident liver (M1)
phagocyte Con A produces proinflammatory cytokines TNF-a, IL-12,
IL-6 and IL-1b that attract CD4? and CD8? T lymphocytes, NK and
NKT cells. These immune cells either directly or through different
soluble mediators induce apoptosis (A) or necrosis (N) of hepatocytes
(H), (gray arrow). IL-33 released from necrotic hepatocytes binds to
the ST2 receptor expressed on immune cells (Th2 and M2) and
converts immune response toward Th2 type, and stimulates secretion
of matrix metalloproteinase and arginase I from alternatively
activated macrophages that promote liver regeneration (Hr) (based
on [47, 60])
92 Immunology in Serbia (2012) 52:89–99
123
ST2 axis is accompanied by enhanced susceptibility to
dominantly T cell-mediated organ-specific autoimmune
diseases.
BALB/c mice are relatively resistant to the induction of T
cell-mediated diabetes by multiple low doses of streptozo-
tocin (MLD-STZ). In BALB/c mice deletion of ST2 mole-
cule leads to enhanced susceptibility to MLD-STZ-induced
disease as evaluated by level of glycemia and glycosuria,
number of infiltrating islet cells and b cell loss [76]. Thus,
ST2-deficient mice develop insulitis and b cell loss by
apoptosis after MLD-STZ induction of disease while there
was minimal apoptosis of beta cells and no infiltrates in the
islets of wild-type, BALB/c mice. Based on these findings,
we considered that the deletion of ST2 molecule and
exclusion of IL-33/ST2 axis alters naturally prevailing Th2
response in BALB/c mice and thus allows development of
autoreactive Th1/Th17 cell-mediated disease such as dia-
betes. We found higher expression of proinflammatory
cytokines TNF-a and IFN-c in pancreatic lymph nodes of
diabetic ST2-deficient mice in the early phase of disease,
and in later phase constantly higher expression of TNF-a.
Additionally, IL-17 was detectable in the later phase of the
disease in ST2-deficient mice, but not in the draining lymph
nodes of the WT mice. Based on these findings in MLD-STZ
diabetes, we suggest that ST2 alters Th1/Th2 balance and
leads to enhanced Th1/Th17 immune response responsible
for the destruction of beta cells and diabetes.
In another model of T cell-mediated autoimmunity,
experimental autoimmune encephalomyelitis, EAE, we
have found that ST2/IL-33 axis has important role in reg-
ulating the encephalitogenic potential of T cells. Although
BALB/c mice are resistant to EAE, ST2 deletion in BALB/
c mice is accompanied by clinically and pathologically
similar expression of EAE as in susceptible C57Bl/6 mice
(unpublished results). Deletions of ST2 gene in BALB/c
mice induced development of highly pathogenic helper
cells in the induction phase of the disease and thereafter
increase influx of these cells in CNS. In spinal cords as well
as in brains of ST2-/- mice at the peak of the disease, we
found higher numbers of CD4? lymphocytes containing
IFN-c, IL-17, TNF-a and GM-CSF, but also myeloid cells
containing IL-33, while in CNS of BALB/c mice at any
time after disease induction, there was negligible number
of inflammatory cells. The importance of ST2 molecule for
EAE development was also shown by adoptive transfer of
immune ST2-/- cells that induced the disease in BALB/c
wild-type mice as in ST2-/- mice. It appears that deletion
of ST2 gene facilitates the development of highly en-
cephalitogenic T helper cells, which can be able to transfer
the disease to WT BALB/c mice. Conversely T cells from
the draining lymph nodes of MOG35-55 immunized WT
BALB/c mice did not induce clinical disease to ST2-/- and
WT recipient. These findings also suggest that ST2
expression on immune cells in CNS is not a crucial factor
that controls inflammation in CNS tissue in EAE. Among
MOG35–55 stimulated mononuclear cells that passively
transferred EAE (ST2-/- cells), we found higher frequency
of cells that contain inflammatory cytokines (IFN-c, IL-17,
TNF-a and GM-CSF) in comparison with cells isolated from
WT mice that were unable to passively induce EAE.
Development of T helper phenotype depends on the function
of APC [77], and our next goal was to determine the effects
of ST2 molecule on APC during EAE. We found that ST2
did not affect the expression of CD80, CD86 and MHCII
markers but affected relative frequencies of different sub-
populations of DC. Inflammatory dendritic cells,
CD11c?CD11b?, that migrate to lymph nodes after initial
Th1 polarizing stimulus produce abundant IL-12 and stim-
ulate IFN-c production in T lymphocytes [78], while
CD11c?CD8? cells act as regulatory cells that suppress
CD4? T lymphocytes [79]. Draining lymph nodes of ST2-/-
BALB/c mice contain higher percentage of inflammatory
type of dendritic cells and lower percentage of
CD11c?CD8? cells. Higher percentage of myeloid cells,
CD11b?, isolated from ST2-/- BALB/c mice contains
proinflammatory cytokines such as IL-1, IL-12, IL-6 and
also IL-33. Similar results were obtained after in vitro cul-
ture of dendritic cells stimulated with TLR agonist. There
were higher amounts of IL-6 and IL-23 and lower amounts
of IL-10 in cell culture supernatants of ST2-/- DC com-
pared to WT derived DC. Based on these findings, we
assume that ST2 deletion alters polarization of APC that
secrete predominantly inflammatory cytokines that leads to
the development of highly encephalitogenic T cells.
Based on our findings, it could be suggested that ST2
gene deletion leads to the production of inflammatory
innate cytokines that induce the development of domi-
nantly inflammatory Th1/Th17 response. Additionally, the
fact that strong proinflammatory type of ST2-/- antigen-
presenting cells is accompanied by higher production of
IL-33 suggests that IL-33 exhibits intracrine inflammatory
role in APC and therefore may also promote inflammation
(illustrated in Fig 2).
IL-33/ST2 axis in anti-tumor immunity
Although there are numerous findings about the role of IL-
33 in inflammation, allergy and autoimmunity, there were
no data about the role of ST2/IL-33 axis in anti-tumor
immunity and tumor growth. Recently, we showed
importance of ST2/IL-33 axis in experimental metastatic
4T1 breast cancer model in mice [80]. ST2-/- mice had
delayed appearance of palpable primary tumor as well as
slower tumor growth and reduced number and size of
metastatic colonies in lungs and livers. ST2 deletion was
Immunology in Serbia (2012) 52:89–99 93
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accompanied by increased number of CD4? and CD8? T
lymphocytes, enhanced cytotoxic activity in vitro of
splenocytes, NK and CD8? T lymphocytes, and also
increased the numbers of IFN-c expressing NK cells. In
contrast, number of IL-10-producing NK cells was higher
in wild-type mice after tumor inoculation, while unde-
tectable in ST2-deficient mice. ST2-/- mice also have
constitutively higher percentages of activated
CD27highCD11bhigh NK cells and CD69?KLRG- NK
cells. In vivo depletion of CD8? or NK cells revealed the
key role for NK cells in enhanced anti-tumor immunity in
ST2-/- mice.
The role of IL-33/ST2 axis on NK cells function is not
fully understood. There are reports of IL-33 to directly
stimulate [32] or indirectly amplify [31] responses of iNKT
and NK cells. However, the IL-33-dependent enhancement
of IFN-c production by these cells always required the
presence of IL-12. Our results appear to be at variance with
Fig. 2 Possible mechanism of IL-33/ST2 axis impact on autoimmune
disorder development. Autoantigen challenge of ST2-/- mice in the
presence of adjuvant (or STZ-induced release of autoantigens, not
shown) induce proinflammatory polarization of antigen-presenting
cells in the draining lymph node. ST2-/- APC after stimulation with
adjuvant produce high amount of proinflammatory cytokines IL-12,
IL-1, IL-6 (see text). These APC also contain high level of IL-33 that
possibly could, by intracrine action, potentiate production of inflam-
matory cytokines. These APC present antigen to naive T cells and
induce differentiation toward Th1 type cells that express T-bet and
produce IL-17, IFN-c, GM-CSF and TNF-a. These inflammatory T
cells pass blood–tissue barrier, enter the target tissue, secrete
inflammatory cytokines and attract other immune cells that secrete
soluble factors that damage tissue. If ST2 molecule is present in APC,
autoantigen induces limited production of inflammatory cytokines and
in these cells production of IL-10 prevails. Thus, these APC polarize
naive T cells toward Th2 type that express GATA-3. Th2 cells
produce minor amount of inflammatory cytokines, do not pass blood–
tissue barrier and therefore tissue damage is not seen
94 Immunology in Serbia (2012) 52:89–99
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these reports [31, 32]. Possible explanation of this dis-
crepancy may be related to in vivo maturation of dendritic
cells in ST2-/- mice and their effect on NK cells [81].
Dendritic cells with mature phenotype appear to be
required for the functional maturation of NK cells [81].
Mayuzumi et al. [82] have recently demonstrated that
conventional myeloid dendritic cells from IL-33 supple-
mented cultures are immature and resistant to phenotypic
and functional maturation. Thus, it could be assumed that
in vivo lack of ST2 signaling may facilitate maturation of
dendritic cells. In fact, we have obtained data indicating
that percentage and number of CD11c?CD80highCD86high
dendritic cells were significantly higher in the local lymph
nodes of ST2-/- tumor-bearing mice compared to WT
mice [80]. Thus, it appears that IL-33/ST2 signaling
facilitates primary tumor progression and metastatic dis-
semination probably affecting cytotoxic activity and cel-
lular makeup of local lymph node and spleen, indicating an
important regulatory role of IL-33/ST2 pathway in NK
physiology and anti-tumor immunity.
It also appears that ST2 deletion affects macrophage
differentiation in tumor-bearing host. Macrophages can be
categorized into two main subsets in parallel with Th1/Th2
dichotomy. M1 macrophages (classically activated) are
induced with IFN-c and characterized as IL-12- and IL-23-
producing cells that exert enhanced cytotoxic activity
against neoplastic cells [83–87]. M1 macrophages kill
tumor cells, secrete high amounts of proinflammatory
cytokines and activate anti-tumor immune response [86,
88], while M2 macrophages play a crucial role in type 2
immune response: promoting angiogenesis, remodeling
and repairing of damaged tissues and also controlling
inflammatory response by downregulation of M1-mediated
functions [86, 89–92]. Since ST2 is constitutively expres-
sed on alternatively activated macrophages [33], we won-
dered whether IL-33/ST2 axis is involved in control of
tumor development through activity of macrophages. We
showed that target disruption of ST2 is associated with
constitutive frequency of alternatively activated (CD206?)
macrophages in the spleen [80].
Thus, IL-33 could be one of the cytokines that influence
modulation of macrophages toward M2 cells, which pro-
duce IL-10 and suppress innate and adaptive anti-tumor
immune responses.
Our recent data (unpublished results) show that exoge-
nously administrated IL-33 enhances primary 4T1 tumor
growth and inhibits innate anti-tumor immunity. It appears
that IL-33 acts as an important amplifier of the develop-
ment of alternatively activated macrophages and also
markedly reduced NK cell cytotoxic activity.
Based on our findings in mouse mammary adenocarci-
noma 4T1 cancer model [80, 93], it could be suggested that
Fig. 3 Scheme of the observed effects of IL-33/ST2 axis on tumor
growth. The effects of endogenous and also exogenous IL-33 in
mammary adenocarcinoma (4T1)-bearing hosts. IL-33 activates T
cells toward Th2 phenotype and generates relatively immature
dendritic cells that do not produce IL-12p70. Immature dendritic
cells induce T-regs that contribute to an immunosuppressive
environment and facilitate metastasis. Subsequently, IL-33/ST2
signaling could upregulate OX40L on dendritic cells leading to
induction of IL-4, and more importantly immunosuppressive IL-10-
and IL-13-producing Th2-cells that promote cancer escape. IL-33/
ST2 signaling could possibly enhance the production of tumor cells
factors with immunosuppressive activity. Also, IL-33 induces IL-10
production in NK cells and decreases their cytotoxicity. In the
absence of ST2, IL-33 produced by epithelial cells and possibly tumor
cells does not lead to the activation of Th2-associated immunosup-
pressive response. Concomitantly, IL-12 produced by classically
activated M1 macrophages leads to the maturation of DC and
induction of strong Th1-polarized immune response followed by IFN-
c production, which activates tumoricidal CTLs and NK cells. These
cells with enhanced cytotoxic activity delay 4T1 tumor growth and
the development of metastases (modified from [80, 93])
Immunology in Serbia (2012) 52:89–99 95
123
the absence of ST2 molecule decreases the immunosup-
pressive Th2-type immune response mediated by IL-33
released from epithelial, endothelial or maybe tumor cells.
Then, IL-12 produced by classically activated M1 macro-
phages promote maturation of DC and consequently strong
Th1/Th17 response that activates tumoricidal NK, NKT
cells and CD8? T cytotoxic lymphocytes (illustrated in
Fig 3). In the wild-type mice, if IL-33 is overexpressed
either endogenously or exogenously, it binds to ST2-posi-
tive Th2-polarized cells and also promotes generation of
relatively immature dendritic cells that could induce T-regs
and therefore facilitate tumor progression and metastasis.
Therapeutic potential of IL-33 and soluble ST2
IL-33 is a dual-role cytokine. It can not only promote but
also reduce inflammation depending on the tissue envi-
ronment [35]. In addition, although firstly described as
Th2-type promoting cytokine, now it is known that IL-33
has pleiotropic effects, it can contribute to development of
Th1-type of immune response as well as enhanced IL-1 and
IL-18 secretion. It implies that IL-33/ST2 axis could be
considered as therapeutic target in different diseases. IL-33
can reduce inflammation depending on the tissue context,
for example, in blood vessel inflammation associated with
atherosclerosis [36]. Unexpectedly, application of IL-33 in
established Th1/Th17 mediated inflammatory conditions
such as collagen-induced arthritis exacerbated the disease
[94]. Besides, in chronic inflammation, addition of IL-33
can promote fibrosis through enhancing the production of
cytokines such as IL-13 and factors secreted by alterna-
tively activated macrophages.
Studies with IL33-/-, ST2-/- mice and application of
different anti-ST2 antibodies in the same model of disease
indicated that absence of IL-33 do not have the same
impact on disease development/attenuation as well as
absence of ST2 molecule [95]. The fundamental mecha-
nisms of the synthesis, processing, releasing and active
form of IL-33 are still poorly defined [25]. In addition, it is
unknown whether is there any other receptor that could
bind IL-33 or weather IL-33 shares ST2 as receptor with
some other cytokine. Better understanding of these pro-
cesses is essential for the future studies of IL-33 as thera-
peutic agent.
Serum level of sST2 molecule is increased in many
inflammatory conditions with anti-inflammatory effects.
Treatment of mice with mixed Th1/Th2 type of inflam-
matory bowel disease with standard anti-TNF therapy led
to attenuation of the disease that was accompanied by
higher sST2/IL-33 ratio in serum [96]. Also, adenovirus-
mediated overexpression of soluble ST2 protects from
LPS-mediated lung injury [97]. The finding that serum
sST2 increases in response to serial injection of IL-33
indicates that ST2 is induced as a negative regulator of IL-
33 [95]. Thus, it seems that modulation of serum level of
sST2 and its relative ratio with IL-33 should be further
explored as potential therapeutic target in many inflam-
matory conditions. To this, we also added the evidence that
ST2 deletion/blocking may enhance anti-tumor immunity
and stimulate NK cell activity. This may have therapeutic
implication in immune response to viruses and experi-
mental immunotherapy of malignances.
Acknowledgments This study is supported by grants ON175069,
ON175071 and ON175103 from Ministry of Education and Science,
Republic of Serbia. We thank Dr. Andrew McKenzie for providing us
ST2 knockout mice and also thank Milan Milojevic for excellent
technical assistance.
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