Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis

Abstract

Ulcerative colitis (UC) is characterized by chronic relapsing intestinal inflammation finally leading to extensive tissue fibrosis and resulting in a stiff colon unable to carry out peristalsis or to resorb fluids. Telocytes, a peculiar type of stromal cells, have been recently identified in the human gastrointestinal tract. Several roles have been proposed for telocytes, including mechanical support, intercellular signalling and modulation of intestinal motility. The aim of the present work was to investigate the presence and distribution of telocytes in colonic specimens from UC patients compared with controls. Archival paraffin-embedded samples of the left colon from UC patients who underwent elective bowel resection and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were identified by CD34 immunohistochemistry. In early fibrotic UC cases, fibrosis affected the muscularis mucosae and submucosa, while the muscularis propria was spared. In advanced fibrotic UC cases, fibrosis extended to affect the muscle layers and the myenteric plexus. Few telocytes were found in the muscularis mucosae and submucosa of both early and advanced fibrotic UC colonic wall. In the muscle layers and myenteric plexus of early fibrotic UC, telocytes were preserved in their distribution. In the muscularis propria of advanced fibrotic UC, the network of telocytes was reduced or even completely absent around smooth muscle bundles and myenteric plexus ganglia, paralleling the loss of the network of interstitial cells of Cajal. In UC, a loss of telocytes accompanies the fibrotic remodelling of the colonic wall and might contribute to colonic dysmotility.

Keywords: CD34; colonic wall; fibrosis; immunohistochemistry; interstitial cells of Cajal; myofibroblasts; telocytes; ulcerative colitis.

Fig. 1

Masson's trichrome-stained sections of colonic wall from patients with ulcerative colitis (UC). Representative microphotographs of sections from UC patients in an early-phase (A, B, D, E, G and H) or an advanced phase (C, F and I) of fibrotic remodelling of the colonic wall are shown. (A–C) Low magnification views of the colonic mucosa and submucosa show the presence of histopathological lesions typical of UC, such as surface epithelial damage, erosions, goblet cell depletion, cryptitis, crypt abscesses, distortion and/or destruction and a marked infiltration of inflammatory and immune cells throughout the muscularis mucosae and submucosa. (D–F) Submucosa. (G–I) Muscle layers and myenteric plexus. In early fibrotic UC cases, fibrosis affects the muscularis mucosae (A and B), while the submucosa is characterized by the presence of areas displaying oedema and a pattern of incoming fibrosis (A and D) and areas of established fibrosis (B and E). Note collagen fibres surrounded by oedema in an area of the submucosa (D), while tightly packed collagen bundles are visible in another area (E). No fibrosis is observed in the muscularis propria (G and H). In advanced fibrotic UC cases, an increased deposition of extracellular matrix is observed in the muscularis mucosae, which appears markedly thickened (C), widespreadly in the submucosal layer (C and F), and also in wide areas of the muscle layers and myenteric plexus (I). Note the accumulation of dense collagen bundles surrounding myenteric ganglia (I). All tissue sections are stained with Masson's trichrome with aniline blue (red colour: cytoplasm; blue colour: collagen). mm: muscularis mucosae; SM: submucosa; CM: circular muscle layer; MP: myenteric plexus; LM: longitudinal muscle layer. Scale bar = 400 μm (A–C), 200 μm (D–F), 100 μm (G–I).

Fig. 2

Muscularis mucosae and submucosa of colonic wall specimens from controls (A, D, G and J), early fibrotic ulcerative colitis (UC) cases (B, E and H) and advanced fibrotic UC cases (C, F and I). (A–C) Double immunofluorescence labelling for CD34 (green) and α-smooth muscle actin (α-SMA, red) with DAPI (blue) counterstain for nuclei. (D–J) CD34 immunoperoxidase labelling (brownish-red) with haematoxylin counterstain. (A) In the muscularis mucosae of control colonic wall, numerous telocytes are present among smooth muscle bundles. Smooth muscle cells are α-SMA-positive. Inset: Higher magnification view of a telocyte located in the muscularis mucosae displaying a slender nucleated body and long, thin and varicose processes. (B and C) In the muscularis mucosae of early fibrotic UC (B) and advanced fibrotic UC (C) colonic wall, very few or no telocytes are observed among smooth muscle bundles. CD34 immunopositivity can be observed only in vessels. Note that the muscularis mucosae appears markedly thickened and infiltrated by inflammatory and immune cells. (D, G and J) In the submucosa of control samples, telocytes form an abundant network and surround vessels and ganglia (arrowheads in G and J). Insets in D and G: Representative higher magnification views of submucosal telocytes with long and varicose telopodes. (E, F, H and I) In the submucosa of early fibrotic UC (E and H) and advanced fibrotic UC (F and I) specimens, telocytes are severely reduced or even completely undetectable. Note a telocyte dispersed in the submucosal matrix and surrounded by some inflammatory cells (inset in E). Few or no telocytes can be observed around submucosal ganglia surrounded by the inflammatory infiltrate (arrowheads in H and I). Scale bar = 100 μm (A–F), 50 μm (G–I), 30 μm (J). (K) Results of quantitative analysis of telocytes in the muscularis mucosae and submucosa of controls (n = 10), early fibrotic UC (n = 7) and advanced fibrotic UC (n = 5) patients. Data are mean values and SEM. *P < 0.05 versus control. mm: muscularis mucosae; SM: submucosa.

Fig. 3

Submucosa and submucosal border of the circular muscle layer of colonic wall specimens from controls (A and D), early fibrotic ulcerative colitis (UC) cases (B and E) and advanced fibrotic UC cases (C and F). (A–C) Double immunofluorescence labelling for CD34 (green) and the pan-endothelial cell marker CD31 (red) with DAPI (blue) counterstain for nuclei. Telocytes are CD34-positive and CD31-negative, while vascular endothelial cells are CD34/CD31-double-positive (arrows). (D–F) CD34 immunoperoxidase labelling (brownish-red) with haematoxylin counterstain. Numerous telocytes are present throughout the submucosa of control colonic wall (A and D), while they are very few or absent in the submucosa of both early fibrotic (B and E) and advanced fibrotic (C and F) UC cases. Inset in A: Higher magnification view of a submucosal blood vessel encircled by telocyte prolongations. In control specimens, telocytes form an almost continuous layer at the submucosal border of the muscularis propria (arrowheads in A and D). Inset in D: Higher magnification view of a submucosal telocyte bordering the circular muscle layer with two long and varicose processes. The layer of telocytes at the submucosal border of the muscularis propria is discontinuous in early fibrotic UC cases (arrowheads in B and E) or completely undetectable in advanced fibrotic UC cases (arrowheads in C and F). SM: submucosa; CM: circular muscle layer. Scale bar = 100 μm (A–F).

Fig. 4

Submucosa of colonic wall specimens from controls (A), early fibrotic ulcerative colitis (UC) cases (B) and advanced fibrotic UC cases (C). (A–C) Double immunofluorescence labelling for CD34 (green) and α-smooth muscle actin (α-SMA, red) with DAPI (blue) counterstain for nuclei. (A) In the submucosa of control specimens, a network of CD34-positive telocytes is visible, while very few α-SMA-positive spindle-shaped cells (myofibroblasts) are present. Inset in A: Higher magnification view of a telocyte with two long varicose processes. In the submucosa of both early fibrotic (B) and advanced fibrotic (C) UC cases, there are few or no telocytes, whereas α-SMA-positive myofibroblasts are numerous. As expected, in all colonic sections α-SMA immunopositivity is also observed in vascular wall pericytes and smooth muscle cells (arrows). Insets in B and C: Representative higher magnification views of submucosal myofibroblasts. mm: muscularis mucosae; SM: submucosa. Scale bar = 100 μm (A–C). (D) Results of quantitative analysis of submucosal myofibroblasts in controls (n = 10), early fibrotic UC (n = 7) and advanced fibrotic UC (n = 5) patients. Data are mean values and SEM. *P < 0.05 versus control.

Fig. 5

Muscularis propria of colonic wall specimens from controls (A, D and G), early fibrotic ulcerative colitis (UC) cases (B, E and H) and advanced fibrotic UC cases (C, F and I). (A–C) CD34 immunoperoxidase labelling (brownish-red) with haematoxylin counterstain. (D–I) Immunofluorescence labelling for CD34 (green) with DAPI (blue) counterstain for nuclei. (A, D and G) In control colonic sections, telocytes are numerous among smooth muscle bundles of the circular and longitudinal muscle layers, form a network around the myenteric ganglia (arrowheads) and are also present in the interganglionic region of the myenteric plexus. Insets in A and G: Higher magnification views of telocytes enveloping ganglia with their long and varicose processes. (B, E and H) In both muscle layers and myenteric plexus region of early fibrotic UC cases, telocyte distribution is similar to that observed in control samples. Note the abundant network of telocytes around myenteric ganglia (arrowheads). (C, F and I) In advanced fibrotic UC cases, very few telocytes are visible in fibrotic areas of muscle layers, and the telocyte network is discontinuous or even almost absent around ganglia (arrowheads) and in the interganglionic region of the myenteric plexus. Scale bar = 100 μm (A–F), 50 μm (G–I). (J) Results of quantitative analysis of telocytes in the circular muscle layer, myenteric plexus region and longitudinal muscle layer of controls (n = 10), early fibrotic UC (n = 7) and advanced fibrotic UC (n = 5) patients. Data are mean values and SEM. *P < 0.05 versus control. CM: circular muscle layer; MP: myenteric plexus; LM: longitudinal muscle layer.

Fig. 6

Muscularis propria of colonic wall specimens from controls (A–C and G–I) and advanced fibrotic ulcerative colitis (UC) cases (D–F and J–L). (A–L) Double immunofluorescence labelling for CD34 (green) and c-kit/CD117 (red) with DAPI (blue) counterstain for nuclei. Telocytes are CD34-positive/c-kit-negative, whereas interstitial cells of Cajal (ICC) are c-kit-positive/CD34-negative. (A–C) In muscle layers of control colonic sections, telocytes and ICC form interconnected networks among smooth muscle bundles. (D–F) In muscle layers of advanced fibrotic UC cases, very few telocytes and ICC can be observed. Representative microphotographs of the circular muscle layer are shown. (G–I) Note the abundant networks of telocytes and ICC around ganglia (arrow in I) and in the interganglionic region (asterisk in I) of the myenteric plexus of control colonic wall. Inset in I: Higher magnification view of an ICC surrounded by telocyte prolongations. (J–L) In advanced fibrotic UC cases, both telocytes and ICC are scarce around myenteric ganglia (arrow in L) and in the interganglionic region (asterisk in L). CM: circular muscle layer; MP: myenteric plexus. Scale bar = 50 μm (A–L).

Fig. 7

Results of quantitative analysis of interstitial cells of Cajal (ICC) in the circular muscle layer, myenteric plexus region and longitudinal muscle layer of controls (n = 10), early fibrotic UC (n = 7) and advanced fibrotic UC (n = 5) patients. Data are mean values and SEM. *P < 0.05 versus control. CM: circular muscle layer; MP: myenteric plexus; LM: longitudinal muscle layer.