Telocytes in Crohn's disease

Abstract

Crohn's disease (CD) is a relapsing chronic inflammatory disorder that may involve all the gastrointestinal tract with a prevalence of terminal ileum. Intestinal lesions have a characteristic discontinuous and segmental distribution and may affect all layers of the gut wall. Telocytes (TC), a peculiar type of stromal cells, have been recently identified in a variety of tissues and organs, including gastrointestinal tract of humans and mammals. Several roles have been proposed for TC, including mechanical support, spatial relationships with different cell types, intercellular signalling and modulation of intestinal motility. The aim of our study was to investigate the presence and distribution of TC in disease-affected and -unaffected ileal specimens from CD patients compared with controls. TC were identified by CD34/PDGFRα immunohistochemistry. In affected CD specimens TC disappeared, particularly where fibrosis and architectural derangement of the intestinal wall were observed. In the thickened muscularis mucosae and submucosa, few TC entrapped in the fibrotic extracellular matrix were found. A discontinuous network of TC was present around smooth muscle bundles, ganglia and enteric strands in the altered muscularis propria. At the myenteric plexus, the loss of TC network was paralleled by the loss of interstitial cells of Cajal network. In the unaffected CD specimens, TC were preserved in their distribution. Our results suggest that in CD the loss of TC might have important pathophysiological implications contributing to the architectural derangement of the intestinal wall and gut dysmotility. Further functional studies are necessary to better clarify the role of TC loss in CD pathophysiology.

Keywords: CD34; Crohn's disease; fibrosis; ileum; immunohistochemistry; interstitial cells of Cajal; telocytes.

Figure 1

(A–F) Control ileal specimens. (A and E) Double immunofluorescence labelling for CD34 (green) and CD31 (red). In A, nuclei are counterstained with DAPI. (B–D and F) CD34 immunoperoxidase labelling with haematoxylin counterstain. (A) Mucosa. Note a telocyte (TC) near a glandular crypt (arrow). (B) TC are numerous in the muscularis mucosae and submucosa. (C) Muscularis mucosae. TC are mostly oriented parallel to each other and show a slender-nucleated body and two long processes (arrows). Inset: CD34 immunofluorescence labelling with DAPI counterstain. At higher magnification view, TC varicose processes (telopodes) are identifiable. (D) Submucosa. TC have a roundish body with two or three processes. Note a ganglia of the submucosal plexus surrounded by TC (arrows). (E) Submucosa. TC are particularly concentrated around large blood vessels, where they closely encircle the adventitial layer (arrows), and some TC are located among vascular smooth muscle cells within the vessel wall. Endothelial cells are CD34/CD31 double-positive, while TC are CD34-positive and CD31-negative. (F) Submucosa. TC are located around lymphatic vessels (arrow). Inset: Note the TC processes surrounding the discontinous basal lamina of a lymphatic vessel. mm: muscularis mucosae; SM: submucosa; BV: blood vessel; LV: lymphatic vessel. Scale bars are indicated in each panel.

Figure 2

(A–H) Control ileal specimens. (A and F) Double immunofluorescence labelling for CD34 (green) and CD31 (red). (G) Double immunofluorescence labelling for CD34 (green) and c-kit (red). (B–E and H) CD34 immunoperoxidase labelling with haematoxylin counterstain. (A and B) Submucosa and circular muscle layer. Telocytes (TC) form a continuous layer bordering the circular muscle layer (arrows). (C) Muscularis propria. TC form a network around smooth muscle fibres. Inset: At higher magnification view, intramuscular TC display a roundish or oval body and three or four varicose processes. (D) Muscularis propria. TC are embedded in the connective tissue between muscle bundles. (E and F) Myenteric plexus. TC form a network enveloping the ganglia (arrows) and the enteric strands in intergangliar region (arrowheads). TC processes are intermingled with ganglion cells. (G) Myenteric plexus. TC and interstitial cells of Cajal (ICC) encircle together the myenteric plexus. (H) Subserosa. TC are visible around vessels, nerves and among adipocytes. SM: submucosa; CM: circular muscle layer; MP: myenteric plexus; S: subserosa; LM: longitudinal muscle layer. Scale bars are indicated in each panel.

Figure 3

(A–G and I) Affected ileal specimens from Crohn’s disease (CD) patients. (H) Control ileal specimens. (A, E, F and I) CD34 immunoperoxidase labelling with haematoxylin counterstain. (B–D) Masson’s trichrome staining. (G and H) Double immunofluorescence labelling for CD34 (green) and α-smooth muscle actin (α-SMA) (red). (A and B) Mucosa and submucosa. Severely damaged tissue with a haemorrhagic ulcer. (A) Telocytes (TC) are not visible, while many CD34-positive microvessels are present (arrow). Inset: CD34/CD31 double immunofluorescence labelling with DAPI counterstain. At higher magnification view, many CD34/CD31-positive microvessels are evident. (B) The inflammation extends to the muscularis mucosae and submucosa, with a pattern of incoming fibrosis. (C) Mucosa, muscularis mucosae and submucosa. The thickened and fibrotic muscularis mucosae extends to the circular muscle layer, replacing the normal submucosa. (D) Muscularis mucosae. Muscle cells are disarranged and separated by fibrosis. (E and F) Muscularis mucosae. Most of the TC disappear (E) and the few remaining ones are located among smooth muscle cells (F). Inset: Higher magnification view displaying TC around smooth muscle bundles. (G and H) Submucosa. (G) In affected CD specimens, CD34/α-SMA double immunolabelling highlights the loss of TC and the presence of many α-SMA-positive spindle-shaped myofibroblasts. (H) In control specimens, many CD34-positive TC are present, while myofibroblasts are not detectable. In both CD and control specimens, vascular pericytes display α-SMA positivity (arrowheads). (I) Submucosa. Few or no TC are present around submucosal ganglia (arrows). u: ulcer; mm: muscularis mucosae; SM: submucosa. Scale bars are indicated in each panel.

Figure 4

(A–F) Affected ileal specimens from Crohn’s disease (CD) patients. (A–D) Immunoperoxidase labelling for CD34 (A–C) and α-smooth muscle actin (α-SMA) (D) with haematoxylin counterstain. (E and F) Double immunofluorescence labelling for CD34 (green) and CD31 or α-SMA (red) with DAPI counterstain. (A) Submucosa. Telocytes (TC) encircle blood vessels and enlarged lymphatic vessels. (B) A reactive lymphoid aggregate appears entirely encircled by TC (arrows). (C and D) A lymphoid aggregate, completely embedded in the fibrotic extracellular matrix (C, asterisk), is surrounded by few TC and display many newly formed microvessels (C, arrow). The presence of fibrosis (D, asterisk) is confirmed by numerous α-SMA-positive myofibroblasts (D, arrow). (E and F) CD34/CD31 and CD34/α-SMA double immunolabelling confirms the absence of TC and the presence of numerous microvessels and myofibroblasts. LV: lymphatic vessel. Scale bars are indicated in each panel.

Figure 5

(A–F) Affected ileal specimens from Crohn’s disease (CD) patients. (A–E) CD34 immunoperoxidase labelling with haematoxylin counterstain. (F) Double immunofluorescence labelling for CD34 (green) and c-kit (red). (A and B) Muscularis propria. Telocytes (TC) maintain their morphology and distribution around and along smooth muscle fibres in a spared area of muscularis propria (A, left side; B, upper side), while they are not present in the close damaged area (A, right side; B, lower side). (C–F) Myenteric plexus. (C) Irregular distribution of ganglia (asterisks) between the circular and longitudinal muscle layers. (D and E) The TC network around ganglia (arrows) and enteric strands in intergangliar regions is discontinuous or even completely absent. (F) A discontinuous network of CD34-positive TC and c-kit-positive ICC is present around myenteric plexus ganglia. TC and ICC are almost completely absent in muscle layers, while many CD34-positive microvessels (arrows) are evident. MP: myenteric plexus. Scale bars are indicated in each panel.

Figure 6

(A–G) Affected ileal specimens from Crohn’s disease (CD) patients. (A) Masson’s trichrome staining. (B–G) Immunoperoxidase labelling for α-smooth muscle actin (α-SMA) (B and G) and CD34 (C–F) with haematoxylin counterstain. (A) At the border with the subserosa, longitudinal muscle bundles are separated by inflammatory infiltrate and fibrous connective tissue. In consecutive immunostained sections, many α-SMA-positive myofibroblasts are visible (B) while CD34-positive telocytes (TC) are absent (C). (D and E) In areas of the subserosa displaying inflammatory infiltration and incoming fibrosis, TC are not visible around vessels (D), but they are still present around nerves (E). (F and G) In areas of the subserosa with settled fibrosis, TC are rare or completely absent (F), while numerous α-SMA-positive myofibroblasts are observed (G). LM: longitudinal muscle layer; LV: lymphatic vessel; S: subserosa. Scale bars are indicated in each panel.

Figure 7

(A–C) Control ileal specimens. (D–F) Affected ileal specimens from Crohn’s disease (CD) patients. (A–F) Double immunofluorescence labelling for CD34 (green) and platelet-derived growth factor receptor α (PDGFRα) (red). In the ileal wall, all CD34-positive telocytes (TC) are also PDGFRα-positive. (A–C) In control specimens, TC form a broad network surrounding the myenteric plexus. (D–F) In severely damaged areas of affected CD specimens, the TC network around the myenteric plexus is discontinuous or even absent. MP: myenteric plexus. Scale bars are indicated.