A novel mast cell-dependent allergic peritonitis model

Abstract

Typical murine models of allergic inflammation are induced by the combination of ovalbumin and aluminum hydroxide. However, accumulating evidence indicates that, in models of asthma and atopic dermatitis, allergic inflammation can be generated in the absence of aluminum hydroxide. Moreover, co-administration of Staphylococcus aureus enterotoxin B with ovalbumin can enhance inflammation. The objective of this study was to establish a rapid and mast cell-dependent murine model of allergic inflammation by inducing allergic peritonitis using ovalbumin and S. aureus enterotoxin B. Allergic peritonitis was induced in C57BL/6 mice by subcutaneous sensitization and intraperitoneal challenge with ovalbumin and S. aureus enterotoxin B. Disease characteristics were assessed by flow cytometry, enzyme-linked immunosorbent assay (ELISA), trypan blue exclusion and colorimetric assays. The time-course of the allergic peritonitis revealed a peak of peritoneal inflammation 48 h after challenge, as assessed by total cells and eosinophil counts. The decrease of cell numbers started 96 h post-challenge, with complete clearance within 168 h. Moreover, significantly higher levels of tryptase and increased vascular permeability were found 30 min following challenge. Allergic inflammation induction by ovalbumin and S. aureus enterotoxin B was impaired in mast cell-deficient mice and partially restored by mice reconstitution with bone marrow-derived mast cells, indicating the mast cell role in this model. We present a novel model of allergic peritonitis that is mast cell-dependent, simple and robust. Moreover, the use of S. aureus enterotoxin B better resembles human allergic inflammation, which is known to be characterized by the colonization of S. aureus.

Keywords: allergic inflammation; allergic peritonitis; animal models; eosinophils; mast cells.

FIGURE 1

Time–course of inflammation in the ovalbumin/Staphylococcus aureus enterotoxin B activator protein (OVA/SEB AP). (a) OVA/SEB AP was carried out in 8–9‐week‐old C57BL/6 wild‐type (WT) female mice. Inflammation was measured, at different time‐points, by (b) peritoneal total cells (trypan blue), (c) eosinophils, (d) mast cells (MCs), (e) macrophages (FC analysis) cell numbers and (f) sCD48 levels [enzyme‐linked immunosorbent assay (ELISA)]. Data are the mean ± standard error of the mean (SEM), n = 4–5 mice per group; *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 2

Cytokine levels from total cells in the peritoneal lavage of C57BL/6 mice following the activator protein (AP) model. (a) Interleukin (IL)‐4, (b) IL‐17A and (c) interferon (IFN)‐γ levels in the peritoneal lavage of phosphate‐buffered saline (PBS)‐ or ovalbumin/Staphylococcus aureus enterotoxin B (OVA/SEB)‐challenged mice 48 h after challenge. Data are the mean ± standard error of the mean (SEM), n = 4 mice/group, *p < 0.05

FIGURE 3

Ovalbumin and Staphylococcus aureus enterotoxin B (OVA/SEB)‐specific immunoglobulin (Ig)E production in the OVA/SEB activator protein (AP) model. (a) OVA‐specific IgE levels in the sera of wild‐type (WT) OVA/SEB‐ or phosphate‐buffered saline (PBS)‐challenged mice 48 h following challenge. (b) Tumor necrosis factor (TNF)‐α release from serum‐sensitized, OVA‐ or SEB‐activated WT bone marrow‐derived mast cells. (BMMCs). Data are the mean ± standard error of the mean (SEM), n = 3; *p < 0.05

FIGURE 4

Vascular permeability and soluble mediators release in the peritoneal lavage of C57BL/6 mice following the activator protein (AP) model. (a) Tryptase, (b) tumor necrosis factor (TNF)‐α levels in peritoneal lavage and (c) vascular permeability in the peritoneal cavity of C57BL/6 mice 30 min following challenge as measured by fluorescein isothiocyanate (FITC)‐dextran levels in peritoneal lavage. Data are the mean ± standard error of the mean (SEM), n = 5–7 mice/group; *p < 0.05, **p < 0.01

FIGURE 5

Ovalbumin/Staphylococcus aureus enterotoxin B activator protein (OVA/SEB AP) model in mast cell (MC)‐deficient mice. OVA/SEB AP was carried out in 8–9‐week‐old C57BL wild‐type (WT) and KitW‐sh/W‐sh (Sash) female mice. Inflammation was measured at the 48 h time‐point by (a) peritoneal total cells (trypan blue), (b) eosinophils, (c) mast cells (MCs), (d) macrophages [fluorescence activated cell sorter (FACS) analysis] cell numbers, n = 6–8 mice per group and (e) sCD48 levels [enzyme‐linked immunosorbent assay (ELISA)], n = 6–8 mice per group. Data are the mean ± standard error of the mean (SEM); *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 6

Ovalbumin/Staphylococcus aureus enterotoxin B activator protein (OVA/SEB AP) inflammation after bone marrow‐derived mast cells (BMMC) reconstitution (overshoot) in mast cell (MC)‐deficient mice. (a) Total cell and (b) eosinophils in peritoneum lavage of wild‐type (WT) mice or KitW‐sh/W‐sh (Sash) mice, with or without reconstitution of WT bone marrow‐derived mast cells BMMCs 48 h after challenge. Data are the mean ± standard error of the mean (SEM), n = 4–6 mice/group; *p < 0.05