Strictures in Crohn's disease are characterised by an accumulation of mast cells colocalised with laminin but not with fibronectin or vitronectin

Abstract

Background/aims: Intestinal fibrosis and stricture formation is an unresolved problem in Crohn's disease. The aim of this study was to investigate whether mast cells accumulate in these tissues and whether their localisation is associated with extracellular matrix components.

Methods: Mast cells were visualised by immunohistochemical staining of the mast cell specific proteases chymase and tryptase. Their localisation in relation to extracellular matrix components was shown by immunohistochemical double labelling.

Results: In strictures in Crohn's disease, a striking accumulation of mast cells was seen particularly in the hypertrophied and fibrotic muscularis propria, with a mean (SEM) mast cell number of 81.3 (14.9) v 1.5 (0.9)/mm(2) in normal bowel (p<0.0005). All mast cells in the muscularis propria were colocalised with patches of laminin. In contrast, in the submucosa, laminin was exclusively found in the basal lamina of blood vessels where many adherent mast cells were seen. No colocalisation of mast cells was found with fibronectin or vitronectin.

Conclusions: The large accumulation of mast cells in the muscle layer of strictured bowel suggests a functional role for these cells in the hypertrophic and fibrotic response of the smooth muscle cells. The colocalisation with laminin indicates a mechanism of interaction between smooth muscle cells and mast cells that may be important in the role of mast cells in the process of fibrosis.

Figure 1

Distribution of mast cells in normal colonic tissue. Mast cells were visualised by immunohistochemical staining for mast cell tryptase (pink colour) with the biotin-avidin-peroxidase system. The mucosa, submucosa, and muscularis propria are shown. Mast cells are found in the mucosa and submucosa. Hardly any mast cells are detectable in the muscularis propria. Of the two mast cells seen in the muscularis propria (arrows), one is located in a connective tissue septum reaching from the submucosa into the muscle layer. Original magnification ×50.

Figure 2

Distribution of mast cells in the muscularis propria in colonic strictures in Crohn's disease. (A) Mast cells are shown by immunohistochemical peroxidase staining for mast cell tryptase (red colour). An impressive accumulation of mast cells is found in the inner layer of the muscularis propria (lower part of the picture) beneath the submucosa (upper half of the picture). Original magnification ×100. (B) Localisation of mast cells in the inner (top two thirds of the picture) and outer (bottom) layer of the muscularis propria in an ileal stricture in Crohn's disease. Glucose oxidase staining for chymase was used to detect mast cells (dark blue colour). The highest numbers of mast cells are seen in the inner layer beneath the submucosa and in the outer layer of the muscularis propria. Original magnification ×100.

Figure 3

Numbers of mast cells in colonic strictures in Crohn's disease compared with normal colon. Mast cells were detected by staining for tryptase and chymase. Mast cell numbers were determined in five adjacent microscope fields in the mucosa, submucosa, and muscularis propria at 200 × magnification and normalised to 1 mm². Mast cell numbers for the three different bowel compartments of strictured colon of Crohn's disease were compared with those of the respective compartments of normal colon: †p<0.005, ‡p<0.0005,**p<0.01.

Figure 4

Numbers of mast cells in ileal strictures in Crohn's disease and in normal ileum. Mast cells were detected by staining for tryptase and chymase. Mast cell numbers were determined in five adjacent microscope fields in the mucosa, submucosa, and muscularis propria at 200 × magnification and normalised to 1 mm². Mast cell numbers for the three different bowel compartments of the ileum of Crohn's disease were compared with those of the respective compartments of normal ileum: †p<0.005, **p<0.01.

Figure 5

Numbers of mast cells in the muscularis propria of involved but non-strictured bowel compared with strictured bowel segments of five individual patients. Mast cells were visualised by staining for tryptase, and numbers represent cells counted in five adjacent fields at 200 × magnification normalised to 1 mm².

Figure 6

Numbers of mast cells in the muscularis propria in the colon in ulcerative colitis and sigmoid diverticulitis and in normal colon. Mast cells were detected by staining for tryptase and chymase and counted in five adjacent fields and normalised to 1 mm². Only numbers of mast cells in sigma diverticulitis were significantly different from those in normal colon: *p<0.05, †p<0.005.

Figure 7

Localisation of laminin and mast cells in strictured and normal colon. Immunohistochemical double staining was used to demonstrate laminin (red colour) and mast cell chymase (grey colour). (A) Shown is the muscularis propria of a fibrotic stricture of the colon in Crohn's disease. Patches of laminin (red colour) are colocalised with mast cells (grey colour). Original magnification ×200. (B) In the muscularis propria, mast cells (grey colour) are located in the centre of laminin deposits (red colour). Original magnification ×400. (C) Distribution of laminin and mast cells in the submucosa in the same fibrotic stricture. Laminin is mainly located in the basal lamina of blood vessels where many adherent mast cells are found. A small part of the inner layer of the muscularis propria is shown in the left lower corner. Original magnification ×100. (D) Localisation of laminin in the normal colon. Laminin is found in the basal lamina of blood vessels in the submucosa (top of the picture). There are no laminin deposits and no mast cells in the muscle layer (lower part of the picture). Original magnification ×10\.

Figure 8

Deposition of fibronectin and vitronectin in the muscularis propria in colonic strictures in Crohn's disease. (A) Fibronectin (red colour) is diffusely distributed in the muscle layer. There is no strict colocalisation with mast cells (grey colour). Original magnification× 200. (B) Vitronectin (red colour) is spotted in the muscle layer. No colocalisation with mast cells (grey colour) is seen. Original magnification ×200.