The cytokine interleukin-33 mediates anaphylactic shock

Abstract

Anaphylactic shock is characterized by elevated immunoglobulin-E (IgE) antibodies that signal via the high affinity Fc epsilon receptor (Fc epsilonRI) to release inflammatory mediators. Here we report that the novel cytokine interleukin-33 (IL-33) potently induces anaphylactic shock in mice and is associated with the symptom in humans. IL-33 is a new member of the IL-1 family and the ligand for the orphan receptor ST2. In humans, the levels of IL-33 are substantially elevated in the blood of atopic patients during anaphylactic shock, and in inflamed skin tissue of atopic dermatitis patients. In murine experimental atopic models, IL-33 induced antigen-independent passive cutaneous and systemic anaphylaxis, in a T cell-independent, mast cell-dependent manner. In vitro, IL-33 directly induced degranulation, strong eicosanoid and cytokine production in IgE-sensitized mast cells. The molecular mechanisms triggering these responses include the activation of phospholipase D1 and sphingosine kinase1 to mediate calcium mobilization, Nuclear factor-kappaB activation, cytokine and eicosanoid secretion, and degranulation. This report therefore reveals a hitherto unrecognized pathophysiological role of IL-33 and suggests that IL-33 may be a potential therapeutic target for anaphylaxis, a disease of considerable unmet medical need.

Fig. 1.

IL-33 in atopic patients during anaphylaxis and inflammation. (A) Total IgE levels in healthy donors, atopic controls, and atopic patients with anaphylactic shock. (B) IL-33 levels in healthy donors, atopic controls, and atopic patients with anaphylactic shock. Data are mean ± SD, n = 5 for each group. *P < 0.01 compared to healthy. (C) Total RNA was extracted from skin biopsy samples of healthy donors and skin lesions of atopic dermatitis patients. IL-33 mRNA was determined by quantitative polymerase chain reaction (Q-PCR). Data are mean ± SD, n = 7, *P < 0.01 compared to healthy skin. (D) Immune staining with anti-IL-33 antibody or control IgG. Pictures are representative of skin biopsy samples of seven healthy and seven dermatitis donors.

Fig. 2.

Passive cutaneous anaphylaxis (PCA). PCA was induced as described in Materials and Methods. (A–D) Mice were treated as follows: Treatment 1, injected with PBS alone; 2, sensitized with IgE alone; 3, challenged with IL-33 alone; 4, sensitized with IgE and challenged with DNP/HSA; 5, sensitized with IgE and challenged with IL-33; and 6, sensitized with IgE and challenged with DNP/HSA+IL-33. (A, B) In wild-type (WT) mice, IL-33 alone did not induce PCA (group 3) but induced PCA and mast cell degranulation in IgE-sensitized mice (group 5). IL-33 enhanced antigen (DNP-HSA) induced PCA and mast cell degranulation (group 6). (C, D) IL-33 induced PCA and mast cell degranulation in RAG1^−/− mice as efficiently as in WT mice. (E, F) Mice were treated as follows: WT sensitized with IgE and challenged with PBS (WT-IgE+PBS), WT sensitized with IgE and challenged with antigen (WT-IgE+DNP-HSA), WT sensitized with IgE and challenged with IL-33 (WT-IgE+IL-33); ST2^−/− mice sensitized with IgE and challenged with PBS (ST2ko-IgE+PBS), ST2^−/− sensitized with IgE and challenged with antigen (ST2ko-IgE+DNP-HSA), ST2^−/− sensitized with IgE and challenged with IL-33 (ST2ko-IgE+IL-33). IL-33 induced PCA in WT but not in ST2^−/− mice. Panels B, D, and F are toluidine blue stained samples and degranulated mast cells are indicated by arrows. Results in A–F are means ± SD, n = 6. *P < 0.01, compared with control mice injected with PBS alone or IgE+PBS.

Fig. 3.

Systemic anaphylactic shock in mice. For systemic anaphylactic shock in wild-type mice, mice were injected i.v. with the various reagents as indicated in the figure and in Materials and Methods (treatments 1–11). (A) Rectal temperature was measured every 10 minutes after challenge. (B) Serum histamine, (C) plasma cytokines and (D) chemokines were examined at 120 minutes after challenge. Data are mean ± SEM, n = 5, *P < 0.01 compared to group 1, +P < 0.01 compared with group 5. (E) Lung sections from mice injected i.v. with the various reagents as indicated in the figure and in Materials and Methods (treatments 1–11), were stained with hematoxylin and eosin and examined under light microscopy. The pictures are representative of five mice per group. For full treatment details, please refer to the method, “Induction and Measurement of Anaphylactic Shock.” (F) To test the specificity of IL-33–induced systemic anaphylactic shock, ST2^−/− mice were injected i.v. with the various reagents as indicated in the figure and in Materials and Methods (treatments 1–6), data are mean ± SEM, n = 5, *P < 0.01 compared with group 1.

Fig. 4.

IL-33 triggers mast cell degranulation and proinflammatory mediators in an ST2-dependent manner. (A) Wild-type mast cells were sensitized with IgE and β-hexosaminidase release was measured after 30 minutes of stimulation with increasing concentration of IL-33. (B) Wild-type mast cells (nonsensitized) were stimulated with increasing concentrations of IL-33, and β-hexosaminidase release was measured after 30 minutes of stimulation. (C) Wild-type and ST2^−/− mast cells were sensitized with IgE and β-hexosaminidase release was measured after 30 minutes of stimulation with: 10 ng/ml of IL-33; or 1 μg/ml of antigen DNP-HSA or a combination of both triggers. PGD₂ (D), LTB₄ (E), cytokines (F), and chemokines (G) production was determined 24 hours after 10 ng/ml of IL-33 stimulation or from wild-type and ST2^−/− mast cells. Results are means ± SD, n = 3, and from three independent experiments, *P < 0.01 compared to basal level. (H) Cell surface expression levels of FcεRI and ST2 on nonsensitized and IgE-sensitized murine mast cells. Results are representative from three independent experiments.

Fig. 5.

Effect of IL-33 on ovalbumin (OVA) sensitized mice. (A) OVA induces similar levels of IgE in wild-type (WT), mast cell–deficient (MC^−/−) and ST2-null (ST2^−/−) mice. (B–E) Mice were treated as follows: treatment 1, WT mice sensitized with PBS and challenged with IL-33; 2, WT mice sensitized with OVA and challenged with PBS; 3, WT mice sensitized with OVA and challenged with IL-33; 4, MC^−/− mice sensitized with PBS and challenged with IL-33; 5, MC^−/− mice sensitized with OVA and challenged with PBS; 6, MC^−/− mice sensitized with OVA and challenged with IL-33; 7, ST2^−/− mice sensitized with PBS and challenged with IL-33; 8, ST2^−/− mice sensitized with OVA and challenged with PBS; 9, ST2^−/− mice sensitized with OVA and challenged with IL-33. (B) Rectal temperature was measured every 10 minutes after IL-33 challenge (C) Serum histamine, (D) plasma cytokines were examined at 120 minutes after challenge. Data are mean ± SEM, n = 5, *P < 0.01 compared to group 1, +P < 0.01 compared with group 5. (E) Lung sections from mice euthanized at 120 minutes after challenge were stained with hematoxylin and eosin and examined under light microscopy. Pictures are representative of five mice per group.