Divergent Effects of Acute and Prolonged Interleukin 33 Exposure on Mast Cell IgE-Mediated Functions

Abstract

Background: Epithelial cytokines, including IL-33 and Thymic stromal lymphopoietin (TSLP), have attracted interest because of their roles in chronic allergic inflammation-related conditions such as asthma. Mast cells are one of the major targets of IL-33, to which they respond by secreting cytokines. Most studies performed thus far have investigated the acute effects of IL-33 on mast cells. In the current study, we investigated how acute vs. prolonged exposure of mast cells to IL-33 and TSLP affects mediator synthesis and IgE-mediated activation. Methods: Human lung mast cells (HLMCs), cord blood-derived mast cells (CBMCs), and the ROSA mast cell line were used for this study. Receptor expression and the levels of mediators were measured after treatment with IL-33 and/or TSLP. Results: IL-33 induced the release of cytokines. Prolonged exposure to IL-33 increased while TSLP reduced intracellular levels of tryptase. Acute IL-33 treatment strongly potentiated IgE-mediated activation. In contrast, 4 days of exposure to IL-33 decreased IgE-mediated activation, an effect that was accompanied by a reduction in FcεRI expression. Conclusion: We show that IL-33 plays dual roles in mast cells, in which its acute effects include cytokine release and the potentiation of IgE-mediated degranulation, whereas prolonged exposure to IL-33 reduces IgE-mediated activation. We conclude that mast cells act quickly in response to the alarmin IL-33 to initiate an acute inflammatory response, whereas extended exposure to IL-33 during prolonged inflammation reduces IgE-mediated responses. This negative feedback effect suggests the presence of a novel regulatory pathway that modulates IgE-mediated human mast cell responses.

Keywords: FcεRI; IL-33; IgE; TSLP; mast cells.

Figure 1

Expression of receptors for IL-33 (ST2) and TSLP (TSLP-R/IL7R) on various human mast cells. Surface expression of TSLP-R (A), IL7R (B), and ST2 (C) on ROSA (A–F), CBMC (A–C), and HLMCs (A–C) was measured by flow cytometry. Representative histograms in which dotted lines represent the respective isotype controls are shown for different cell lines to the left, and quantification of the data is shown to the right (A–C). Mast cells were treated with 10 ng/ml IL-33, TSLP or a combination or both repeatedly for 4 days; thereafter, surface expression of TSLP-R (D), IL7R (E), and ST2 (F) was measured by flow cytometry. The median fluorescent intensity (MFI) of the receptors was normalized to the respective isotype control. Data shown were pooled from three independent experiments, n = 3–6.

Figure 2

Histamine release induced by prolonged exposure to IL-33. ROSA cells (A,B) or CBMCs (C,D) were treated with 10 ng/ml IL-33, TSLP or a combination of both for 1 h (A,C) or treated repeatedly (every day) for 4 days (B,D). Thereafter, supernatants were collected, and histamine was measured. Data shown were pooled from 3 to 5 independent experiments, n = 6–10.

Figure 3

Prostaglandin D₂ and cytokine release in response to IL-33. CBMCs were treated for 1 h (A–C) or 24 h (D–I) with 10 ng/ml IL-33 or 2 μM A23187, and supernatants were collected. PGD₂ (A–C) was measured using ELISA, and cytokines were measured using Luminex assays (D–I). The CBMCs shown in (B) were pretreated with 10 ng/ml IL-4 and 5 ng/ml IL-3 for 4 days. Data shown were pooled from 5 to 9 independent experiments, n = 5–9.

Figure 4

Changes in mediator storage after 4 days of treatment with IL-33 and TSLP. ROSA cells (A,E) or CBMCs (B–D,F) were repeatedly treated every day for 4 days with 10 ng/ml IL-33, TSLP or a combination of both; thereafter, the levels of the proteases tryptase (A,B), chymase (C), and CPA3 (D) were measured by intracellular flow cytometry staining, and total histamine content (E,F) was measured by a histamine release test kit. The MFI of protease expression was normalized to the respective isotype control. (A) a representative of three independent experiments, n = 3. Data shown were pooled from 5 (B), 2 (C,D), 3 (E), and 6 (F) independent experiments, n = 4–12.

Figure 5

Effect of IL-33 on mast cell degranulation by FcεRI crosslinking. CBMCs were treated with 10 ng/ml IL-33 for 1 h (A,C) or repeatedly every day for 4 days (B,D); thereafter, they were stimulated various concentrations of anti-IgE. Degranulation was measured by the detection of surface CD63 with flow cytometry (A,B) or as histamine release (C,D). Data shown were pooled from 3 independent experiments, n = 3.

Figure 6

Decrease in FcεRI surface expression by IL-33. ROSA cells (A,D), CBMCs (B,E), or HLMCs (C) were repeatedly treated every day for 4 days with 10 ng/ml IL-33, TSLP or a combination of both. In addition, IL-4 was added on day 0 (A,B) or 4 days prior to the addition of IL-33 and TSLP (D,E) to upregulate the FcεRI receptor. IL-4 was not added to HLMCs (C). Surface FcεRIα receptor expression was measured by flow cytometry; representative histograms are shown in (A–C) and quantification of the expression is shown below. The MFI of FcεRI was normalized to that of the isotype control. Data shown were pooled from 3 to 5 independent experiments, n = 6–10.

Figure 7

Decrease in mRNA expression of the FcεRI receptor by IL-33. CBMCs were repeatedly treated every day for 4 days with 10 ng/ml IL-33, TSLP or a combination of both. In addition, IL-4 was added on day 0. The mRNA expression level of the different subunits of the FcεRI receptor were quantified using qPCR (A–C). Expression was normalized to that of the housekeeping gene GAPDH and thereafter to the unstimulated control. Data shown were pooled from six independent experiments, n = 6.