Activation of Eosinophils and Mast Cells in Functional Dyspepsia: an Ultrastructural Evaluation

Abstract

We recently identified mucosal mast cell and eosinophil hyperplasia in association with a duodenal impaired barrier function in functional dyspepsia (FD). We aimed to further describe the implication of these immune cells by assessing their activation state at the ultrastructural level and by evaluating the association between impaired epithelial integrity and immune activation. Duodenal biopsies were obtained from 24 FD patients and 37 healthy controls. The ultrastructure of mast cells and eosinophils was analyzed by transmission electron microscopy. Transepithelial electrical resistance and paracellular permeability were measured to evaluate epithelial barrier function. The type of degranulation in eosinophils and mast cells was piecemeal. Eosinophils displayed higher degree of degranulation in FD patients than in controls (p < 0.0001). Quantification revealed a decreased granular density in eosinophils of FD patients (p < 0.0001). The degree of degranulation in mast cells was similar in both groups. However, a more heterogeneous profile was found in the FD group (p < 0.0001). No association between epithelial integrity and the number and activation state of mucosal eosinophils and mast cells was found. We demonstrated ultrastructural changes in degranulation state of eosinophils and mast cells, suggesting that eosinophil and mast cell activation play a role in the pathophysiology of FD.

Figure 1

Evaluation of low-grade inflammation. Duodenal biopsy samples from healthy volunteers (control) and patients with FD were stained for identification of eosinophils using anti-human MBP (A,B) and mast cells using anti-human tryptase (C,D) antibodies. Cells positive for MBP (n = 31 for controls and n = 20 for patients with FD) (A) and tryptase (n = 36 for controls and n = 20 for patients with FD) (C) were counted per mm² in at least seven non-overlapping fields per subject. Data are mean ± SEM. Representative images of MBP (B) and tryptase (D) immunohistochemistry in mucosal biopsy specimens obtained from a control (left) and a patient with FD (right). Scale bar: 20 µm. **p < 0.01, ***p < 0.001. FD, functional dyspepsia; MBP, major basic protein.

Figure 2

Ultrastructural evaluation of mucosal eosinophils. Duodenal biopsy samples from healthy volunteers (control) and patients with FD were used to evaluate degranulation of eosinophils using transmission electron microscopy. Visual analysis of the presence (n = 25 for controls and n = 17 for patients with FD) (A) and degree (n = 15 for controls and n = 14 for patients with FD) (B) of degranulation in eosinophils. Quantitative analysis of eosinophilic granular density evaluated as the pixel intensity distribution (C) and the mean intensity per granule (D) (n = 25 for controls and n = 15 for patients with FD). (E) Representative transmission electron micrographs of eosinophils in mucosal biopsy specimens obtained from a control (left) and a patient with FD (right). Lower panels show representative cytoplasmic granules from each group. Note higher loss of granular content in granules from FD group (arrows). Bars indicate magnification (top panels: 2 µm; low panels: 0.5 µm) **p < 0.01, ***p < 0.001. FD, functional dyspepsia; N, nuclei; PMD, piecemeal degranulation.

Figure 3

Comparison of visual and quantitative analysis of eosinophilic degranulation. (A) The mean intensity per granule was compared between the three groups: no (n = 13), low (n = 19) and high (n = 8) degranulation. (B) Correlation between the mean intensity per granule and the degree of eosinophilic degranulation: no (n = 13), low (n = 19) and high (n = 8).

Figure 4

Ultrastructural evaluation of mucosal mast cells. Duodenal biopsy samples from healthy volunteers (control) and patients with FD were used to evaluate degranulation of mast cells using transmission electron microscopy. Degree of degranulation (A) and complexity of the granule content (B) in mast cells (n = 28 for controls and n = 19 for patients with FD). (C) Representative transmission electron micrographs of mast cells in mucosal biopsy specimens obtained from a control (left) and a patient with FD (right). Lower panels show representative cytoplasmic granules from each group. Degranulation is present in cells from both groups (arrows). Bars indicate magnification (top panels: 2 µm; low panels: 0.2 µm). **p < 0.01. FD, functional dyspepsia; N, nuclei; PMD, piecemeal degranulation.