Role of mast cells in chronic stress induced colonic epithelial barrier dysfunction in the rat

Abstract

Background and aims: Stress may be an important factor in exacerbating inflammatory bowel disease but the underlying mechanism is unclear. Defective epithelial barrier function may allow uptake of luminal antigens that stimulate an immune/inflammatory response. Here, we examined the effect of chronic stress on colonic permeability and the participation of mast cells in this response.

Methods: Mast cell deficient Ws/Ws rats and +/+ littermate controls were submitted to water avoidance stress or sham stress (one hour/day) for five days. Colonic epithelial permeability to a model macromolecular antigen, horseradish peroxidase, was measured in Ussing chambers. Epithelial and mast cell morphology was studied by light and electron microscopy.

Results: Chronic stress significantly increased macromolecular flux and caused epithelial mitochondrial swelling in +/+ rats, but not in Ws/Ws rats, compared with non-stressed controls. Stress increased the number of mucosal mast cells and the proportion of cells showing signs of activation in +/+ rats. No mast cells or ultrastructural abnormalities of the epithelium were present in Ws/Ws rats. Increased permeability in +/+ rats persisted for 72 hours after stress cessation.

Conclusions: Chronic stress causes an epithelial barrier defect and epithelial mitochondrial damage, in parallel with mucosal mast cell hyperplasia and activation. The study provides further support for an important role for mast cells in stress induced colonic mucosal pathophysiology.

Figure 1

Effect of chronic stress on colonic physiology and macromolecule permeability. Mast cell deficient rats (Ws/Ws) and their normal mast cell containing littermates (+/+) were submitted to sham stress or water avoidance stress for five consecutive days. (A) Baseline short circuit current (Isc), (B) conductance (G), and (C) horseradish peroxidase (HRP) flux studied in Ussing chambers six hours after the final sham/stress session. Bars represent mean (SEM); n=6 rats/group, with 2-4 tissues averaged per rat. **p<0.01 v all other groups.

Figure 2

Effect of chronic stress on horseradish peroxidase (HRP) transport and epithelial mitochondria. Mast cell deficient rats (Ws/Ws; left) and their normal mast cell containing littermates (+/+; right) were submitted to water avoidance stress (top photomicrographs) or to sham stress (bottom photomicrographs) for five consecutive days. (A) Photomicrograph showing an HRP filled endosome (large arrowhead) in a colonocyte of a stressed Ws/Ws rat. Paracellular passage of HRP and mitochondrial abnormalities were not observed in Ws/Ws rats. (B) Larger and more numerous HRP filled endosomes (arrowheads) as well as HRP within tight junctions and paracellular spaces between colonocytes (arrows) were observed in the colon of stressed +/+ rats, as shown in this photomicrograph. Epithelial damage is demonstrated by swollen mitochondria with loss of cristae. (C, D) Colonocytes with small endosomal uptake of HRP (arrowheads) and normal mitochondria from sham stressed Ws/Ws (C) and +/+ rats (D), respectively. Mitochondrial surface area was significantly increased in stressed +/+ rats compared with the three other groups (table 1). Magnification: ×3000. Each photomicrograph is representative of four rats/group and 15 sections/colonic tissue.

Figure 3

Effect of chronic stress on mast cells in +/+ rats. (A) A non-activated mast cell (MC) in the mucosa of a sham stressed +/+ rat showing numerous homogeneous electron dense granules. (B) A mast cell (MC) with piecemeal-type degranulation. The mast cell is in close apposition to an emptied nerve terminal (N) in the mucosa of a stressed +/+ rat, and shows loss of intragranular density without fusion of intergranular membranes, retained oval granules in the cytoplasm, and absence of extruded granules contents or granule membranes, indicative of piecemeal-type degranulation. (C) A mast cell (MC) in the mucosa of a stressed +/+ rat showing enlarged granules with solubilisation of intragranular contents, fusion of intergranular membranes (arrows), and absence of extruded material, indicative of a mixed anaphylactic-piecemeal degranulation pattern. Magnification: A-C, ×3000.