See 1 citation in J Leukoc Biol 2008 by Hudson:
J Leukoc Biol. 2008 Sep;84(3):631-43. doi: 10.1189/jlb.1207830. Epub
2008 Jun 13.
Induction of IL-33 expression and activity in central nervous system glia.
- 1
- Department of Neurology and Interest Group in Neuro-Immune Interactions, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
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-1beta 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.
Fig. 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]. ♦, Significant difference versus the PAM3Cys group.
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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 40× and 63×). (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].
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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.
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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.
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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.
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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.
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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 × 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.
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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-α (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-α (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).
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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.
J Leukoc Biol. 2008 Sep;84(3):631-643.
Fig. 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.
J Leukoc Biol. 2008 Sep;84(3):631-643.
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