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Originally published online as doi:10.1189/jlb.1207830 on June 13, 2008

Published online before print June 13, 2008
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(Journal of Leukocyte Biology. 2008;84:631-643.)
© 2008 by Society for Leukocyte Biology

Induction of IL-33 expression and activity in central nervous system glia

Chad A. Hudson*, George P. Christophi*, Ross C. Gruber*, Joel R. Wilmore*, David A. Lawrence{dagger} and Paul T. Massa*,1

* Department of Neurology and Interest Group in Neuro-Immune Interactions, SUNY Upstate Medical University, Syracuse, New York, USA; and
{dagger} Clinical and Experimental Endocrinology and Immunology, Wadsworth Center, Albany, New York, USA

1 Correspondence: Department of Neurology, Upstate Medical University, State University of New York, 750 East Adams St., Syracuse, NY 13210, USA. E-mail: massap{at}upstate.edu

ABSTRACT

IL-33 is a novel member of the IL-1 cytokine family and a potent inducer of type 2 immunity, as mast cells and Th2 CD4+ T cells respond to IL-33 with the induction of type 2 cytokines such as IL-13. IL-33 mRNA levels are extremely high in the CNS, and CNS glia possess both subunits of the IL-33R, yet whether IL-33 is produced by and affects CNS glia has not been studied. Here, we demonstrate that pathogen-associated molecular patterns (PAMPs) significantly increase IL-33 mRNA and protein expression in CNS glia. Interestingly, IL-33 was localized to the nucleus of astrocytes. Further, CNS glial and astrocyte-enriched cultures treated with a PAMP followed by an ATP pulse had significantly higher levels of supernatant IL-1β and IL-33 than cultures receiving any single treatment (PAMP or ATP). Supernatants from PAMP + ATP-treated glia induced the secretion of IL-6, IL-13, and MCP-1 from the MC/9 mast cell line in a manner similar to exogenous recombinant IL-33. Further, IL-33 levels and activity were increased in the brains of mice infected with the neurotropic virus Theiler’s murine encephalomyelitis virus. IL-33 also had direct effects on CNS glia, as IL-33 induced various innate immune effectors in CNS glia, and this induction was greatly amplified by IL-33-stimulated mast cells. In conclusion, these results implicate IL-33-producing astrocytes as a potentially critical regulator of innate immune responses in the CNS.

Key Words: IL-1 family cytokines • IL-13 • astrocyte • pathogen-associated molecular pattern

INTRODUCTION

IL-33 is a novel member of the IL-1 family of cytokines and has been linked to the induction of type 2 immune responses [1 ]. Administration of exogenous IL-33 in vivo leads to pronounced splenomegaly and eosinophilia, increased mucous production, and hypertrophy of the digestive and respiratory tracts. These pathological changes occur in conjunction with increases in type 2 cytokines such as IL-4, IL-5, and IL-13 and Th2-related Igs such as IgE [1 ]. IL-33 appears to exert its activities on primarily mast cells and Th2-polarized CD4+ T cells, as both of these cell types are activated by IL-33 in a MyD88- and NF-{kappa}B-dependent manner to secrete type 2 cytokines such as IL-6 and IL-13 [1 2 3 ]. Further, IL-33 can increase the survival and adhesion to fibronectin of human mast cells [4 ] and has been shown to be chemotactic for Th2-polarized T cells [5 ].

Recently, Humphreys et al. [6 ] reported that IL-33 plays an important role in the defense against nematode parasites. IL-33 was up-regulated in the intestine after Trichuris muris infection, and the administration of exogenous IL-33 led to a marked reduction in pathogen burden in mice [6 ]. IL-33 has also been linked with the archetypal Th2 inflammatory disease, asthma. The expression of IL-33 mRNA is very high in the lung [1 ], and in a murine model of asthma, allergic airway inflammation (AAI), the expression of IL-33, and the soluble and membrane-bound forms of a subunit of the IL-33R [named ST2 and ST2 ligand (ST2L), respectively] are induced after OVA challenge [2 ]. Further, splenocytes from AAI-afflicted mice respond to IL-33 with a greater induction of Th2 cytokines than unafflicted mice [2 ]. This induction as well as the induction of NF-{kappa}B by IL-33 can be blocked by the addition of soluble ST2, suggesting that the soluble form of ST2 functions as an inhibitor of IL-33/ST2L signaling [2 ]. The latter finding was recently supported by evidence from models that implicate IL-33 in cardioprotection and the inhibition of athlerosclerosis [7 , 8 ].

Interestingly, the report by Humphreys et al. [6 ] also indicated that IL-33 had T cell-independent functions after T. muris infection on intestinal epithelium, suggesting that epithelial cells could be activated by IL-33. Astrocytes are nonhematopoietic epithelial-like cells of the CNS and are known to express both subunits of the IL-33R, ST2L and IL-1R accessory protein (IL-1Rap) [1 , 9 , 10 ]. The CNS has been found to have extremely high expression of IL-33 at the mRNA level, as the brain and spinal cords are the organs with the highest expression of IL-33 in the body [1 ]. Hence, CNS glia, especially astrocytes, might be potent promoters of IL-33-induced activities.

IL-33 is also believed to act as a transcription factor. It has been shown that IL-33 is localized to the nucleus of endothelial cells and bound to chromatin. Thus, it has been proposed that like the proinflammatory cytokines IL-1{alpha} and high-mobility group box 1, IL-33 acts intracellularly as a transcription factor and extracellularly as a NF-{kappa}B-inducing cytokine [11 ]. This report also demonstrated that IL-33 is up-regulated in chronically inflamed tissues such as the intestines from patients suffering from Crohn’s disease and the synovium of rheumatoid arthritis patients [11 ], suggesting that IL-33 might act in a proinflammatory manner. This suggestion is supported by reports indicating that IL-33 contributes to not only pathogen elimination but also intestinal inflammation in the T. muris model [6 ] and that IL-33 mediates antigen-induced hypernociception (inflammatory pain) [12 ].

Caspase-1, also known as IL-1β-converting enzyme, is responsible for the cleavage of the pro-forms of certain IL-1 family cytokines such as the proinflammatory IL-1β and the IFN-{gamma}-inducing IL-18 [13 ]. IL-33 has also been shown to be cleaved by caspase-1 in vitro [1 ], although the in vivo relevance of this has been debated, as the canine, bovine, and porcine orthologs of IL-33 do not possess a caspase-1 cleavage site [11 ]. Interestingly, treatment with a caspase inhibitor has been shown to reduce AAI in mice [14 ], a finding that suggests caspase-1 processing of IL-1 family member cytokines could be integral in asthma pathogenesis.

The activation of caspase-1 can be mediated through the triggering of the P2X7 receptor (a cell-surface ATP receptor) by ATP. The activation of the P2X7 receptor results in K+ efflux and subsequent activation of caspase-1 via the N-terminal effector domain, a central nucleotide-binding domain, leucine-rich repeat, and pyrin 3 inflammasome, a multisubunit complex that upon activation, leads to the cleavage of procaspase-1 [15 , 16 ]. Secretion of IL-1β and IL-18 via this ATP-driven pathway is reliant on the prior induction of the transcriptional and translational synthesis of the pro-forms of the IL-1 family members [15 ]. In experimental paradigms, the induction of the pro-forms of these cytokines is usually mediated by TLR stimulation by pathogen-associated molecular patterns (PAMPs) such as LPS and dsRNA [16 , 17 ]. As such, it has been demonstrated that in macrophages, LPS and ATP are required to stimulate secretion of IL-1β and IL-18 [15 ]. Further, the sequential treatment of LPS and ATP in microglia also results in the secretion of IL-1{alpha} [18 ], indicating that this paradigm also induces the secretion of IL-1 family cytokines that do not have a caspase-1 cleavage site. The secretion of IL-1{alpha} has been found to be caspase-1-dependent and not a result of cell lysis [19 ]. In fact, it was found that caspase-1 activity regulates the secretion of a variety of unconventionally secreted proteins that lack a caspase-1 site, including IL-1{alpha} [19 ]. Thus, stimulation by a PAMP and ATP (or another inflammasome activator) is required for maximal secretion of caspase-1, processed and unprocessed IL-1 family cytokines.

In this report, we show that IL-33 is inducible by inflammatory stimuli in astrocytes and can directly induce innate type 2 immune effectors and proinflammatory cytokines in CNS glia. Further, an ATP pulse after PAMP treatment induced substantial secretion of not only IL-1β in mixed glial and purified astrocyte cultures but also IL-33. Moreover, the supernatants from the glia that received the PAMP + ATP treatment possessed an IL-33-like activity, as these supernatants induced significantly more IL-13, IL-6, and MCP-1 in the mouse mast cell line MC/9 than supernatants from glia that received PAMP or ATP alone. In addition to a likely IL-33 autocrine loop in astrocytes, bioactive IL-33 from astrocytes may have additional, indirect effects via mast cells, as supernatants from IL-33-treated (exogenous-recombinant or glial-derived) MC/9 cells contained substantial amounts of IL-13 that could amplify glial IL-33-induced type 2 innate responses. The consequences of IL-33 activity in the CNS may thus represent a key factor in CNS inflammatory diseases.

MATERIALS AND METHODS

Mice
Eight-day-old BALB/c and C3FeLe.B6-a.a mice were produced from breeding pairs obtained from National Cancer Institute (Frederick, MD, USA) and Jackson Laboratories (Bar Harbor, ME, USA), respectively, and used to produce mixed CNS glial cultures. All animal procedures described in this study were approved by the Committee for the Humane Use of Animals at Upstate Medical University (Syracuse, NY, USA).

Reagents
Salmonella minnesota re595 LPS (Sigma-Aldrich, St. Louis, MO, USA) and tripalmitoyl-S-glyceryl cysteine (PAM3Cys; EMC Microcollections, Tubingen, Germany) were used at 1 µg/ml, and polyinosinic:polycytidylic acid (dsRNA; Amersham Pharmacia, Piscataway, NJ, USA) was used at 20 µg/ml in all glial experiments. ATP (Sigma-Aldrich) was used at 3 mM in glial experiments. For the direct treatment of mast cells, ATP was used at 1.5 mM after experiencing glial treatment conditions. IL-33 (R&D Systems, Minneapolis, MN, USA) was used at 10 ng/ml (unless otherwise noted), IL-13 (R&D Systems) was used at 2 and 10 ng/ml, and IL-1β (R&D Systems) was used at 1–10 ng/ml.

Glial cultures
Mixed glial cultures were produced from brains of 8-day-old mice as described previously [20 ]. Astrocyte-enriched cultures were produced as described previously [21 ]. For analysis of the induction of IL-33 at the mRNA level, glial cultures were treated with the PAMPs LPS, PAM3Cys, and dsRNA and the cytokine IL-1β for 24 h. A subset of the glia treated with PAM3Cys also received IFN-β. For inducing maximal secretion of IL-1 family cytokines, glial or astrocyte cultures were primed for 8 h at 37°C with the PAMPs LPS, PAM3Cys, and dsRNA. Supernatants were then collected (these supernatants are referred to as prepulse supernatants in the MC/9 experiments), and the glia were washed with 1x PBS to remove TLR agonists prior to the addition of the pulse treatment. Glia were then pulsed with 3 mM ATP for 30 min at 37°C in serum-free phenol red-free DMEM. After 30 min, the pulsed supernatants were collected and frozen. For the mast cell supernatant experiment, glial cultures were treated with recombinant cytokines or supernatant from IL-33-treated MC/9 cells (supernatants diluted 1:1 in DMEM/10% FBS) for 48 h, and supernatants and cell lysates were then collected.

Mast cell cultures
The mouse mast cell line MC/9 was plated at 5 x 105 cells per ml and treated for 24 h, after which, plates were centrifuged at 1200 rpm for 10 min, and supernatant was collected. For experiments in which MC/9 cells were treated with glial supernatants, the supernatants DMEM and FBS were combined to obtain a final concentration of 50% supernatant, 40% DMEM, and 10% FBS

Theiler’s murine encephalomyelitis virus (TMEV) infection and brain extracts
Ten-day-old C3FeLe.B6-a.a mice were infected intracerebrally with 1 x 106 PFU/ml of the BeAN strain of TMEV. Three days postinfection, the mice were killed, and half of the brain was snap-frozen. Later, frozen brains were homogenized in 1 ml PBS and then centrifuged at 13,000 rpm for 10 min at 4°C on a benchtop centrifuge. The extract supernatant was then diluted 1:5 in DMEM + 10% FBS and filtered with a 0.45-µm syringe filter unit (Corning, Corning, NY, USA). Extract supernatant was then diluted 1:1 in DMEM + 10% FBS, and mast cells were then treated as above. MC/9 cultures treated with uninfected and TMEV-infected brain extract supernatants showed no signs of reduced viability compared with MC/9 cells treated with DMEM + 10% FBS.

ELISA
IL-1β, IL-6, and IL-13 were measured using DuoSet ELISA kits (R&D Systems) following the manufacturer’s protocol. MCP-1 was measured using the OptEIA kit (BD Biosciences, San Diego, CA, USA) following the manufacturer’s protocol. IL-18 was measured using antibodies from MBL International (Woburn, MA, USA), and the assay was performed following the manufacturer’s protocol using recombinant IL-18 (rIL-18; MBL International) as the standard. Eotaxin-1 was measured using the BioSource (Invitrogen, Carlsbad, CA, USA) eotaxin Cytoset, following the manufacturer’s protocol. IL-33 was measured via an ELISA that used rat anti-mouse IL-33 (R&D Systems) as the coating antibody at 2 µg/ml and biotinylated goat anti-mouse IL-33 (R&D Systems) as the detection antibody at 1 µg/ml. rIL-33 (R&D Systems) was used as the standard, and a standard curve from 10000 ng/ml to 78.125 ng/ml was used.

Immunohistochemistry
Mixed glial cultures were stimulated with PAMPs for 8 h and then fixed and stained as described previously [20 ]. Briefly, cells were fixed with 0.75% periodate-lysine-paraformaldehyde in PBS for 30 min, permeabilized with 0.25% Triton X-100 in PBS for 15 min, and then blocked with PBS containing 10% normal horse serum for 60 min. IL-33 was stained by overnight incubation with 1 µg/ml goat anti-mouse IL-33 (R&D Systems) followed by the avidin-biotin alkaline phosphatase detection system (Zymed/Invitrogen, Carlsbad, CA, USA). The alkaline phosphatase substrate 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium (BCIP/NBT) was used to produce a blue/violet reaction product (Zymed/Invitrogen). The astrocyte marker glial fibrillary acidic protein (GFAP) was stained by incubating the cells overnight with a 1:500 dilution of rabbit anti-GFAP (2.9 g/l purified rabbit Ig, Dako, Denmark), followed by incubation with a 1:500 dilution of swine anti-rabbit Igs conjugated to HRP (1.3 g/l purified swine Igs, Dako). After rinsing, the cells were incubated with aminoethyl carbazole (AEC; Zymed/Invitrogen) to produce a reddish-brown precipitate. Cells were viewed and photographed by bright-field light microscropy

Arginase activity
Intracellular arginase activity was measured as described previously [21 ].

Real-time RT-PCR
Real-time RT-PCR was performed as described previously [22 ]. The primers used were as follows: IL-33, forward primer 5'-GATGGGAAGAAGGTGATGGGTG-3', reverse primer 5'-TTGTGAAGGACGAAGAAGGC-3'; GAPDH, forward primer 5'-ACCACCATGGAGAAGGC-3', reverse primer 5'-GGCATGGACTGTGGTCATGA-3'.

Statistical analysis
For all multiple group comparisons, one-way ANOVA was performed followed by the Student-Neuman-Keuls method. The Student’s t-test was performed for any individual treatment versus control comparison. P < 0.05 was considered as statistically significant throughout. For the TMEV brain extract experiment, ANOVA on Ranks followed by the Dunn’s test was used as a result of the data failing the normality test.

RESULTS

Induction of IL-33 in CNS glia
Although the expression of IL-33 at the mRNA level has been found to be constitutively high in the CNS, and the induction of IL-33 by IL-1β and TNF-{alpha} has been reported in leukocytes [1 ], the inducibility of IL-33 by inflammatory stimuli in the CNS has not been assessed. To determine this, we observed whether IL-33 mRNA levels were increased after stimulation with the PAMPs dsRNA, LPS, and PAM3Cys, as well as the proinflammatory cytokine IL-1β, which with all three PAMPs significantly increased IL-33 mRNA expression (Fig. 1A ). Further, IL-1β (at 10 ng/ml) induced significantly more IL-33 than all three of the PAMPs. The addition of IFN-β led to a significant inhibition of the induction of IL-33 by PAM3Cys. There were no differences amongst the groups in the control GAPDH mRNA levels (Fig. 1B) .


Figure 1
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  		Figure 1. Induction of IL-33 mRNA in CNS glia. (A) CNS glial cultures were treated with the PAMPs dsRNA, LPS, and PAM3Cys and the proinflammatory cytokine IL-1β for 24 h, and copy numbers of IL-33 mRNA were determined. Some PAM3Cys-treated glia also received IFN-β. (B) GAPDH copy number of CNS glia cultures treated for 24 h with dsRNA, LPS, PAM3Cys, IL-1β, and PAM3Cys + IFN-β. Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method. (A) *, P < 0.05, for that treatment versus control. dsR, dsRNA; L, LPS; P, PAM3Cys [that treatment being significantly greater (P<0.05) than the treatment indicated by that letter]. {diamondsuit}, Significant difference versus the PAM3Cys group.

To identify CNS glia expressing IL-33 protein in glial cultures, IL-33 expression was examined by immunohistochemistry. Although there was little if any IL-33 staining in CNS glial cultures prior to PAMP treatment, following PAMP treatment, IL-33 was expressed exclusively in the nuclei of flattened stellate cells consistent with astrocytic morphology (Fig. 2A ). Cells that expressed a microglial-like morphology did not stain for IL-33 but stained strongly for IL-1β after PAMP treatment (not shown). To corroborate morphological criteria, astrocytes were stained by double immunohistochemistry for IL-33 and the astrocyte marker GFAP. Indeed, IL-33 was found to be expressed almost exclusively in GFAP+ astrocytes. Thus, many double-labeled cells were identified in which IL-33 expression was localized to the nucleus, and GFAP expression was cytosolic (Fig. 2A) . This nuclear localization of IL-33 was similar to that previously reported in endothelial cells [11 ]. PAMP treatment also led to secretion of IL-33, as determined by ELISA, as PAMP treatment led to a significant increase in secreted IL-33 (Fig. 2B) .


Figure 2
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  		Figure 2. Induction of IL-33 in astrocytes. (A) CNS glial cultures were treated with dsRNA and LPS for 8 h and then fixed and immunostained for IL-33 (purple) and the astrocyte marker GFAP (red). The red arrows indicate dsRNA- and LPS-treated astrocytes, and the black arrows indicate untreated astrocytes (at 40x and 63x). (B) Secreted levels of IL-33 from glial cultures treated with LPS, PAM3Cys, or dsRNA. Each graph is the result of an independent experiment that used multiple, independently produced cultures. Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method. *, P < 0.05, for that treatment versus control. P, PAM3Cys [that treatment being significantly greater (P<0.05) than the treatment indicated by that letter].

Induction of IL-1 family cytokines by PAMP + ATP stimulation
As the nuclear staining suggested that most of the IL-33 induced by PAMP stimulation remained in the cell, specifically the nucleus, it was hypothesized that sequential treatment of a PAMP and then ATP might lead to the release of this intracellular store, as shown for IL-1β, IL-18, and IL-1{alpha}. Mixed glial cultures were treated for 8 h with various PAMPs (LPS, PAM3Cys, and dsRNA), and after these prepulse supernatants were collected, the cells were washed and pulsed with ATP for 30 min. A description of the different glial supernatants and the nomenclature used hereafter can be found in Table 1 . In agreement with previous studies in other systems, regardless of which PAMP was used to prime the glia, supernatants from glia that received the PAMP priming plus the ATP pulse had significantly greater levels of IL-1β than any other supernatant (Fig. 3A and B ). However, unlike described previously in macrophages, treatment of glia with PAMP alone was sufficient to induce the secretion of IL-1β (in PAMP prepulse and PAMP+media pulse supernatant). Interestingly, there was no evidence of an induction of IL-18 secretion as a result of priming plus ATP pulse (IL-18 levels ≤15 pg/ml without any significant differences between treatments).


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  		Table 1. Glial Supernatants for PAMP + ATP Experiments


Figure 3
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  		Figure 3. Glial PAMP + ATP-induced, secreted IL-1β and IL-33. (A and B) Secreted levels of IL-1β in the supernatants from mixed glial cultures. Glia were primed for 8 h with dsRNA (A), LPS (A and B), or PAM3Cys (B), and then supernatants (prepulse supernatants) were collected. Cells were then washed and pulsed with (labeled as the PAMP+ATP) or without (labeled as the PAMP+media) ATP in serum-free media for 30 min, and pulsed supernatants were collected. (C) Secreted levels of IL-33 in the supernatants from mixed glial cultures treated for 8 h with LPS. Cells were then washed and pulsed with ATP for 30 min, and pulsed supernatants were collected. (D) IL-33 immunostaining of glial cultures treated with LPS and LPS + ATP. IL-33 staining (purple) is indicated by the arrows in LPS-treated and LPS + ATP-treated cultures. (A and B) *, P < 0.05, for that treatment versus its respective control (i.e., dsRNA vs. media and dsRNA+ATP vs. ATP). For comparisons within a treatment, statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method; bracket with *, P < 0.05, between the two groups linked by the bracket. (C) Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method; bracket with *, P < 0.05, between the two groups linked by the bracket. (A and B) Results of two independent experiments that used multiple, independently produced cultures; (C) two such experiments.

To ascertain similar effects of these treatments on IL-33 secretion in CNS glia, IL-33 was measured by ELISA in the pulsed supernatants. As for IL-1β, the supernatants from glia that received LPS and ATP stimulation had significantly higher levels of IL-33 than any other treatment (Fig. 3C) . Further evidence suggesting that ATP treatment resulted in the release of IL-33 was obtained via immunostaining, as LPS induced the nuclear expression of IL-33, and nuclear IL-33 immunostaining was lessened after ATP treatment (Fig. 3D) . A similar pattern was also observed with PAM3Cys + ATP and dsRNA + ATP (not shown).

Induction of mast cell cytokines by glial PAMP + ATP-treated supernatants
To analyze the bioactivity of the secreted, glia-derived IL-33, a bioassay for IL-33 was developed in which the mast cell line MC/9 cytokine responded to exogenous rIL-33, as reported previously in primary mast cell cultures [3 , 4 ]. At 10 ng/ml, IL-33 significantly induced IL-13, IL-6, and MCP-1 in the MC/9 cells (Fig. 4A 4B 4C , for IL-13; Fig. 4D , for IL-6; Fig. 4E , for MCP-1). To determine the activity of the glia-derived IL-33, MC/9 cells were treated with medium containing the glial supernatants, which from PAMP + ATP-activated glia, regardless of the PAMP used for priming, were able to induce significantly higher levels of IL-13 secretion by MC/9 cells compared with any other supernatant (pulsed and prepulse; Fig. 4A 4B 4C ). This response was not a result of glial production of IL-13, as all media-containing glial supernatant, regardless of treatment, had levels of IL-13 less than 62.5 pg/ml. Like IL-13, MC/9 secretion of IL-6 and MCP-1 was also significantly higher after receiving PAMP + ATP-activated glial supernatants compared with pulsed supernatants from glia receiving other treatments (dsRNA+ATP depicted in Fig. 4D and 4E ).


Figure 4
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  		Figure 4. IL-33-like bioactivity of PAMP + ATP-treated glial supernatants. Secreted levels of IL-13 from MC/9 cells treated for 24 h with IL-33 or the supernatants (from the pulse step and the prepulse step) from glial PAMP + ATP experiments that used LPS (A), dsRNA (B), or PAM3Cys (C). Secreted levels of IL-6 (D) and MCP-1 (E) from the experiment that used dsRNA are also depicted. IL-33 was used at 10 ng/ml if not otherwise noted. Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method. There is a significant difference between treatments if there is a different letter above the bars representing the two treatments.

Characterization of IL-33-like activity in glial supernatants
The induction of IL-13 by the PAMP prepulse supernatants (which contain the majority, if not all, of the PAMPs, as the prepulse media were removed, and the cells were washed prior to the addition of the ATP pulse) indicates that the PAMPs themselves cannot be responsible for the IL-33-like activity observed in the PAMP + ATP-treated supernatants, as PAMP prepulse supernatant induced much lower levels of IL-13 in MC/9 cells than PAMP + ATP-treated supernatant or rIL-33 (Fig. 4A 4B 4C) . To rule out the possibility that ATP and/or IL-1β, present in high concentrations in the PAMP + ATP-activated glial supernatants, were responsible for the IL-13 secretion induced by PAMP + ATP-activated glial supernatants, MC/9 cells were treated with IL-1β and ATP at concentrations at or above the levels found in the PAMP + ATP-activated glial supernatants (1.5 mM ATP and 1–10 ng/ml IL-1β). IL-1β alone (1 and 10 ng/ml) induced little IL-13, and 1 ng/ml IL-1β only slightly increased the amount of ATP-induced IL-13 secretion by MC/9 cells (Fig. 5A ). Thus, although in human mast cells, IL-1β has been shown to be a potent inducer of IL-13 [23 ], in MC/9 cells, it appeared that IL-1β could not induce considerable amounts of IL-13 and therefore, could not be responsible for the induction of IL-13 seen after treatment with the PAMP + ATP-activated glial supernatants. On the other hand, ATP, added directly to MC/9 cells, increased IL-13 secretion over tenfold and induced almost as much IL-13 as exogenous 10 ng/ml IL-33. Thus, ATP induces IL-13 secretion in MC/9 cells, independent of IL-33 or any other cytokine. This agrees with a previous report indicating that ATP can induce IL-13 in mast cells in a STAT6-dependent manner [24 ]. Nonetheless, the induction of IL-13 by ATP and similar to the level of IL-13 observed after treatment with ATP-treated glial supernatant was not sufficient to explain the levels of IL-13 induced by the LPS + ATP-activated glial supernatant. This indicates that the direct effects of ATP on MC/9 cells could be responsible for the induction observed after treatment with ATP-treated glial supernatant (a supernatant that does not have an increased level of IL-33 compared with controls) but not the induction observed after treatment with the PAMP + ATP-treated glial supernatant.


Figure 5
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  		Figure 5. Characterization of the IL-33-like activity. (A) Secreted levels of IL-13 from MC/9 cells treated for 24 h with IL-33, 1 or 10 ng/ml IL-1β, 1.5 mM ATP, or 1 ng/ml IL-1β + 1.5 mM ATP. (B) Secreted levels of IL-13 from MC/9 cells treated for 24 h with supernatants from ATP-treated and LPS + ATP-activated glia and ATP-treated glia supernatant plus 5 ng/ml IL-33. Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method. (A) There is a significant difference between treatments if there is a different letter above the bars representing the two treatments. (B) Bracket with *, P < 0.05, between the two groups linked by the bracket.

To test if the substantial IL-13-inducing capacity of LPS + ATP-activated glial supernatants on mast cells could be mimicked by the addition of rIL-33 to supernatants from ATP-treated glia, 5 ng/ml rIL-33 was added to ATP-treated glial supernatants, and MC/9 cells were treated with this combination, ATP-treated glial supernatants, or LPS + ATP-activated glial supernatants. Exogenous rIL-33/ATP-treated glial supernatant, like LPS + ATP-activated glial supernatant, induced significantly more IL-13 secretion from MC/9 cells than the supernatant from ATP-treated glia not containing exogenous IL-33 (Fig. 5B) . The IL-33/ATP-treated glial supernatant treatment induced even greater levels of IL-13 production than the LPS + ATP-activated glial supernatants, likely as a result of a higher concentration of IL-33 (Fig. 3C) .

Astrocyte-enriched cultures
As IL-33 mRNA is found primarily in cells of epithelial origin [1 ], and we observe nuclear staining of IL-33 in astrocytes (Fig. 2A) , it was of interest to determine whether the induction of IL-1β and IL-33 observed in mixed glial cultures could be attributed to astrocytes (epithelial-like cells of the CNS). Astrocytes were therefore enriched from mixed glial cultures and treated with dsRNA + ATP. As in mixed glial cultures, although dsRNA alone induced significantly more IL-1β in astrocytes than control medium, the supernatants from dsRNA + ATP-activated astrocytes had significantly more IL-1β (Fig. 6A ) and IL-33 [activity (Fig. 6B) and protein levels (Fig. 6C) ] than supernatants from dsRNA-treated, ATP-treated, or untreated control glia. Further, unlike in mixed glial cultures, dsRNA-treated astrocyte cultures secreted significant amounts of IL-18 following an ATP pulse (Fig. 6D) . The secretion of IL-18 indicates the activation of the inflammasome, as the antibodies used in the IL-18 ELISA (MBL International) only recognize the processed form of IL-18. However, the induction of IL-18 was relatively low (19.00±4.08 pg/ml in dsRNA+ATP-activated supernatants compared with 1311.53±37.31 pg/ml for IL-1β).


Figure 6
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  		Figure 6. Astrocyte-enriched cultures. (A) Secreted levels of IL-1β in the supernatants from astrocyte-enriched cultures. Glia were primed for 8 h with dsRNA. Cells were then washed and pulsed with (labeled as the dsRNA+ATP) or without (labeled as dsRNA+media) ATP in serum-free media for 30 min and pulsed supernatants were collected. (B) Secreted levels of IL-13 from MC/9 cells treated for 24 h with IL-33 or the pulse supernatants from the astrocyte-enriched dsRNA + ATP experiment. (C) Secreted levels of IL-33 in the pulse supernatants from the astrocyte-enriched dsRNA + ATP experiment. (D) Secreted levels of IL-18 in the pulse supernatants from the astrocyte-enriched dsRNA + ATP experiment. Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method. There is a significant difference between treatments if there is a different letter above the bars representing the two treatments.

IL-33 activity in the CNS in vivo
To test if IL-33-like activity is induced in the CNS in vivo, mice were infected intracranially with a neurotropic virus (TMEV) and brains analyzed for IL-33 activity 3 days postinfection. TMEV-infected, but not uninfected brain extracts, induced IL-13 production in the MC/9 cells (Fig. 7A ). To confirm this activity correlated with an induction of IL-33, real-time RT-PCR was performed on uninfected and TMEV-infected brains. It was found that TMEV infection significantly increased the level of IL-33 mRNA in the brains of infected mice (Fig. 7B) , indicating that the increase in IL-13-inducing activity correlated with an increased expression of IL-33. There was no difference in GAPDH between TMEV-infected and uninfected brains (Fig. 7C) .


Figure 7
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  		Figure 7. In vivo relevance. (A) Secreted levels of IL-13 from MC/9 cells treated for 24 h with brain extracts from TMEV-infected and uninfected brains. Results are expressed as pg per 5 x 105 cells/g brain protein minus background MC/9 pg/ml. Statistical significance was measured via ANOVA on Ranks followed by the Dunn’s test; *, treatment significantly greater (P<0.05). (B) IL-33 mRNA copy numbers of TMEV-infected and uninfected brains. Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method; and bracket with *, P < 0.05. (C) GAPDH mRNA copy numbers of TMEV-infected and uninfected brains.

Induction of cytokines and immune effectors by IL-33 in CNS glia
Although it is known that astrocytes possess both subunits of the IL-33R, ST2 and IL-1Rap [10 , 25 ], and we have shown that IL-33 can be produced by astrocytes, it had not been determined if IL-33 could induce gene expression in glia. To determine this, CNS glial cultures were treated with 100 ng/ml IL-33 for 24 h, and the mRNA expression of various innate immune genes, proinflammatory cytokines and type 2 chemokines and effector genes (arginase I), was measured. It was determined that IL-33 significantly induced the expression of arginase I (Fig. 8A ), the type 2 chemokines thymus and activation-regulated chemokine (TARC; CCL17) and eotaxin-1 (CCL11; Fig. 8B and 8C ), and the proinflammatory cytokine TNF-{alpha} (Fig. 8D) . Interestingly, this induction of arginase I, TARC, and eotaxin-1, all STAT6-inducible genes and therefore, usually reliant on IL-4 or IL-13 for induction, occurred without the concomitant induction of IL-13 or IL-4 (Fig. 8E and 8F , respectively).


Figure 8
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  		Figure 8. Induction of innate immune effectors and cytokines by IL-33. To measure induction at the mRNA level, CNS glial cultures were treated for 24 h with 100 ng/ml IL-33, and mRNA copy numbers of arginase I (A), TARC (B), eotaxin-1 (C), TNF-{alpha} (D), IL-13 (E), and IL-4 (F) were measured and normalized to the control GAPDH. To determine if this induction led to protein expression of certain effectors and cytokines, CNS glial cultures were treated for 24 h with 10 ng/ml IL-33 and intracellular arginase activity (G), and secreted levels of IL-6 (H), MCP-1 (I), and TNF-{alpha} (J) were measured. Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method, and a P value is displayed if there is a significant difference (P<0.05).

To verify that the induction of mRNA expression by IL-33 treatment led to increased protein expression at a more physiologically relevant level, CNS glial cultures were treated with 10 ng/ml IL-33 for 24 h, and cell lysates were assayed for arginase activity. It was found that IL-33 significantly induced arginase activity in CNS glia (about twofold; Fig. 8G ). Moreover, 10 ng/ml IL-33 was also able to induce the secretion of cytokines. Secreted levels of IL-6, MCP-1 (CCL2), and TNF-{alpha} (Fig. 8H 8I 8J) were increased significantly by IL-33. Levels of IL-1β, NO, IL-4, and IL-13 were below detectable levels (not shown).

To determine if the supernatants from IL-33-treated MC/9 mast cells could amplify glial IL-33-induced type 2 innate responses and induce IL-13-responsive genes in CNS glia, glial cultures were treated with the supernatants from exogenous, IL-33-treated, glia-derived IL-33 (LPS+ATP-treated glial supernatants) and control-treated MC/9 cells. It was found that exogenous and glia-derived rIL-33-treated MC/9 cell supernatants significantly increased the STAT6-responsive arginase I activity in CNS glial cultures (Fig. 9A ) in a manner similar to corresponding amounts of exogenous IL-13. As in Figure 8 , direct administration of IL-33 to CNS glia also induced arginase activity but at a significantly lower level than what IL-33-treated MC/9 supernatants induced (less than twofold vs. >20-fold). IL-33-treated MC/9 cell supernatants significantly increased secreted levels of the IL-13-responsive chemokine MCP-1 [26 27 28 ] (Fig. 9B) beyond the levels induced by exogenous IL-13. Also, direct administration of IL-33 to CNS glia induced as much MCP-1 as exogenous IL-13, indicating that the IL-13 and the IL-33 present in the MC/9 supernatants play a primary role in the considerable induction of MCP-1 observed after treatment with IL-33-treated MC/9 supernatants. Eotaxin-1 displayed a pattern similar to arginase activity, as exogenous and glia-derived rIL-33-treated MC/9 cell supernatants significantly increased the level of secreted eotaxin-1 in a manner similar to corresponding amounts of exogenous IL-13, and direct administration of IL-33 to CNS glia also induced eotaxin-1 but at a significantly lower level (Fig. 9C) .


Figure 9
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  		Figure 9. Effects of IL-33-treated mast cells on glia. Arginase activity (A), MCP-1 (B), and eotaxin-1 (C) secretion of glial cultures treated for 48 h with exogenous, IL-33-treated (labeled 0.1 or 10 ng/ml IL-33 MC Supt), glia-derived IL-33 (LPS+ATP-activated glial supernatants, labeled LPS+ATP MC Supt) and control-treated MC/9 cell supernatants (MC Supt) or exogenous IL-33 or IL-13. For MCP-1, the background levels of MCP-1, present in MC/9 supernatant treatments, were subtracted from the total amount measured in glial supernatants. Statistical significance was measured via one-way ANOVA, followed by the Student-Neuman-Keuls method. There is a significant difference between treatments if there is a different letter above the bars representing the two treatments.

DISCUSSION

This report investigated the potential role for IL-33 as a factor in CNS innate immunity. It was observed that IL-33 is inducible in CNS astrocytes by PAMPs and that upon induction, IL-33 was localized to the nucleus. Further, like other IL-1 family members, internal stores of IL-33 are released by astrocytes (and nuclear levels of IL-33 are thus depleted) after the administration of ATP. Supernatants from PAMP + ATP-activated glia potently induced the secretion of IL-13 in the mast cell line MC/9. Although a portion of the induction seen is undoubtedly the result of the direct action of ATP on the MC/9 cells, PAMP + ATP-activated supernatants displayed substantially higher levels of IL-33-like activity (levels that induced IL-13 in the ng/ml range) compared with ATP alone. In fact, relative to ATP-treated glial supernatants, LPS + ATP-activated supernatants (which are diluted 1:1 in MC/9-inducing experiments) induced 77% of the amount of IL-13 that the 5 ng/ml exogenous IL-33 + ATP-treated supernatant treatment induced (Fig. 5B) . Hence, the LPS + ATP-activated glial supernatants have approximately the same level of IL-33 biological activity as 5 ng/ml IL-33, a finding that meshes well with the ELISA results depicted in Figure 3C , which indicate that LPS + ATP supernatants contain ~1 ng/ml IL-33.

An important question concerning innate immunity is how cell-specific induction of IL-1 family members may affect the nature of ensuing adaptive immunity. As IL-1β, IL-18, and IL-33 have distinct activities in molding adaptive immunity, cell types that respond to the TLR agonist or other PAMP stimulation with a greater induction of one or the other of these cytokines could cause a pronounced shift in Th and overall immune balance, especially in the local tissue microenvironment. In particular, the present data indicate that IL-33 activities may dominate IL-18 activities in the CNS, and this is likely to skew adaptive immunity accordingly. Although there have been reports linking IL-18 to Th2 immunity [29 ], and IL-18 in combination with IL-3 can induce IL-13 in bone marrow-derived mast cells [30 ], IL-18 will most likely primarily promote Th1 immunity [31 32 33 ]. On the other hand, the effects of IL-33 are thought to be almost entirely pro-Th2, as IL-33 induces IL-13 in mast cells and T cells. Hence, if a particular cell type or organ system makes vastly more IL-33 than IL-18 upon PAMP stimulation, the local immune balance is likely to be shifted toward IL-13 and STAT6-mediated activities. Further, if a substantial release of IL-33 is coupled with extensive secretion of IL-1β, then an asthma-like inflammatory state driven by IL-13 and NF-{kappa}B might arise. IL-33 expression is up-regulated in the inflamed organs in experimental asthma and other inflammatory diseases, implicating IL-33 in inflammatory disease [2 , 11 ]. Thus, it is tempting to speculate that under the appropriate conditions, the CNS might be prone to such IL-13/NF-{kappa}B-driven pathology as a result of the high IL-33:IL-18 ratio observed in mixed glial and astrocyte-enriched cultures. This is supported by recent evidence in the CNS inflammatory disease multiple sclerosis, in which STAT6 is preferentially activated in CNS glia [34 ].

An important result that should be noted is that all three PAMPs induced significant, IL-33-like activity in PAMP + ATP-treated glial supernatants. Moreover, the ability of a PAMP to induce IL-33-like activity was completely independent of the ability of a PAMP to directly induce IL-13 in MC/9 cells. This can be observed from the PAMP prepulse glial supernatants. The prepulse supernatants containing LPS and PAM3Cys induced IL-13 secretion at levels significantly higher than the PAMP plus media and media supernatants from the pulse step (Fig. 4A and 4C) , and prepulse dsRNA-treated glial supernatant did not (Fig. 4B) . These findings are in agreement with LPS and PAM3Cys being able to directly induce significant IL-13 production in MC/9 cells, and dsRNA induces little if any IL-13 (C. A. Hudson, unpublished results). Therefore, direct action of the PAMP itself on MC/9 cells has little effect on the activity observed here. IL-18 and TNF-{alpha}, two other prominent NF-{kappa}B inducers, would also not affect the activity observed, as IL-18 (5 and 50 ng/ml) and TNF-{alpha} (10 and 100 U/ml) induce little to no IL-13 in MC/9 cells (data not shown).

Although in our experimental model, all three PAMPs potently induced IL-33 and its activities, it is likely that there would be more heterogeneity in vivo. Specifically, although future studies will have to be conducted to determine which pathogenic stimuli would induce the appropriate conditions for the IL-1β/IL-33-driven inflammatory state described above, we speculate that PAMPs, including those of neurotropic viruses, which induce primarily NF-{kappa}B, would be likely to induce such an inflammatory state. On the other hand, PAMPs such as viral TLR3 agonists that induce substantial amounts of IFN-β, a potent inhibitor of STAT6 and NF-{kappa}B signaling [35 , 36 ], would most likely be relatively inefficient inducers of IL-33-driven inflammation in vivo. Accordingly, prepulse dsRNA glial supernatants in which IFN-β accumulates [37 ] induce less IL-13 (significant via Student’s t-test) from MC/9 cells than the pulsed dsRNA glial supernatant (labeled dsRNA+media in Fig. 4B ), suggesting that there is an inhibitory factor such as IFN-β present in the prepulse dsRNA supernatant. Further, exogenous IFN-β inhibited the induction of IL-33 by PAM3Cys (Fig. 1A and 1B) , a finding similar to what we have observed with the PAM3Cys-induced, MyD88-dependent genes TNF-{alpha} and arginase I (C. A. Hudson, manuscript submitted). On the other hand, TLR2 agonists, such as PAM3Cys, do not induce IFN-β and are therefore expected to be potent inducers of IL-33-driven inflammation. In fact, it has been demonstrated that PAM3Cys skews adaptive immunity toward Th2 and exacerbates experimental asthma in vivo [38 ]. Thus, particular PAMPs that induce NF-{kappa}B but not IFNs would be expected to be the most potent activators of IL-33-driven responses in the CNS.

Although most attention has been focused on the activities of mast cells in the lungs and skin, there is considerable evidence indicating that mast cells are also important in the CNS. It has been long known that in mammals, including rodents, there are relatively high concentrations of mast cells in the leptomeninges, the cerebral cortex, and the thalamus [39 , 40 ]. Other forebrain regions that have significant amounts of mast cells include the hippocampus and the hypothalamus [40 41 42 ]. These brain mast cells appear to be constitutively present and functional in the CNS. In rats, it was found that thalamic mast cells are responsible for up to 90% of the histamine found in the thalamus and for about half of the amount of histamine in the entire brain [43 ]. Further, pharmacologically induced degranulation of mast cells has been shown to regulate thalamic neuronal activity [44 ] and to induce the hypothalamic-pituitary-adrenocortical axis when hypothalamic mast cells are degranulated [41 ]. Brain resident mast cells have also been found to be important in CNS immunity. In a model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), an increase in the number of degranulated mast cells but not total mast cells, was observed in the brains of afflicted animals [45 ]. Further, in acute EAE, there is evidence of perivascular mast cell activation prior to the onset of clinical symptoms, suggesting that mast cell activation may be required for disease initiation [46 ].

In this report, we also show that IL-33 can directly induce gene expression in CNS glia, suggesting that there might be an IL-33 autocrine loop in CNS glia, especially astrocytes. Similar to what is seen upon IL-33 administration in vivo [1 ] or to mast cells in vitro [3 ], IL-33 induces a type 2 immune response, which is clearly illustrated by the increases in arginase I and the type 2 chemokines MCP-1, TARC, and eotaxin-1 observed in IL-33-treated glia. The lack of induction of type 1 mediators such as NO further demonstrates that IL-33 promotes type 2 immunity. However, in the case of glia, this innate type 2 response occurs without the induction of IL-4 or IL-13. Thus, genes usually thought of as downstream of IL-4 and IL-3 in type 2 immune responses are induced directly by IL-33. Although this can be explained for the type 2 chemokines, as each of their promoters possesses NF-{kappa}B-binding sites [47 48 49 50 ], the extrahepatic promoter for arginase I is not known to have a NF-kB site but instead, a STAT6 site and a C/EBP-β site [51 , 52 ]. MyD88-dependent signaling has been shown to induce C/EBP-β [53 ], suggesting that IL-33 might induce arginase I through C/EBP-β, but other inducers of C/EBP-β, such as IL-10, are only able to synergize with IL-4 or IL-13 in the induction of arginase I and cannot induce arginase I on their own [21 ].

Although IL-33 can induce an innate type 2 response directly in CNS glia without IL-4 or IL-13, this induction pales in comparison with the type 2 response that IL-33 can induce in glia when a source of IL-13 is present. For instance, in the presence of mast cells, the IL-33-driven induction of glial type 2 responses is amplified. In the case of arginase I (Fig. 9A) , this amplification is dramatic. Although IL-33 administered to glia increased arginase activity by only approximately twofold, supernatants from MC/9 cells treated with IL-33 induced arginase activity >25-fold. Similar, but not quite as dramatic, results are also seen for glial MCP-1 and eotaxin-1 secretion (Fig. 9B and 9C) . Thus, mast cells serve as an amplification loop for IL-33-driven innate type 2 responses in CNS glia, likely through IL-13 and STAT6, as diagrammed in Figure 10 . IL-33 activates MyD88-dependent mast cell secretion [1 ] of IL-13, and secreted IL-13 induces the activation of STAT6 in glia leading to increased expression of STAT6-responsive genes, such as arginase I [51 ], eotaxin-1 [47 ], and the MCP-1-inducing transcription factor early growth response-1 [54 ]. Further, for genes such as MCP-1, in which MyD88 signaling (IL-33) and STAT6 (IL-13) can lead to a strong induction, IL-33 might not only play an important role in inducing mast cell IL-13 but could also synergize with IL-13 in the induction of MCP-1 in glia. Figure 9B indicates this might be the case, as IL-33-treated MC/9 supernatants induced greater levels of MCP-1 in CNS glia than IL-33 or IL-13, a finding not observed in the primarily STAT6-driven gene arginase I (Fig. 9A) .


Figure 10
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  		Figure 10. Mechanisms of IL-33 induction of innate type 2 immunity in the CNS. Diagram indicating how inflammatory stimuli can induce IL-33-dependent induction of innate type 2 immunity (bold lines) and how the presence of mast cells could lead to an IL-13-dependent amplification of these processes (dashed lines), including a substantial amplification of arginase I and type 2 chemokines.

As there is a relatively large population of resident mast cells in the CNS (as described above), it would seem that if inflammation in the CNS causes the release of IL-33 in vivo, particularly from astrocytes, it would lead to the secretion of IL-13 by mast cells. This could have profound effects in the CNS, as IL-13 may then in turn act on astrocytes and perhaps other CNS glia to induce STAT6-responsive genes to complete an important, neural-immune circuit (akin to Fig. 10 ). This is supported by in vivo experiments in which animals infected with a neurotropic virus (TMEV), which induces pronounced CNS inflammation, induced IL-33 expression and activity in the brain. As mentioned above, one gene that is highly induced in CNS glia after STAT6 induction is arginase I [21 ]. We have found that like IL-33, arginase I expression is induced in the CNS at 3 days post-TMEV infection (G. P. Christophi, unpublished data). Interestingly, this could induce inflammation, as although peripherally, arginase I is usually thought of mostly as anti-inflammatory as a result of its inhibition of NO production [55 ] and its association with wound healing [56 ], it is known to play a pathogenic role in asthma and airway hyper-responsiveness [57 , 58 ] and has been linked with inflammatory disease in the CNS as well. For instance, the expression of arginase I is increased in the PBMCs of multiple sclerosis patients [22 ]. Arginase I was also found to be the most highly induced gene in the CNS of mice with EAE, and pharmacological inhibition of arginase led to a milder disease course [59 ]. Finally, higher expression of arginase I is correlated with a poor prognosis in our TMEV model [21 ]. In all, it seems quite possible that TMEV-induced arginase I expression and TMEV-induced pathology might be downstream of IL-33 secretion. As such, this and the possible mast cell-dependent mechanisms behind it are current topics of research in our laboratory.

In sum, this report illustrates that IL-33 might play an integral role in CNS innate immunity and suggests that the CNS might be uniquely predisposed to respond to certain pathogenic stimuli in a Th2-like manner. We further speculate that cytokine-mediated communication between mast cells and CNS glia represents a key, neural-immune interaction in CNS inflammatory diseases. In future studies, it will be important to determine whether neutralization of IL-33 in the CNS in vivo can abrogate pathology in inflammatory diseases of the CNS.

ACKNOWLEDGEMENTS

This study was supported in part by research grants from the National Multiple Sclerosis Society (RG2569C5) and National Institutes of Health (NS041593) to P. T. M.

Received December 14, 2007; revised May 12, 2008; accepted May 19, 2008.

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