A loss of telocytes accompanies fibrosis of multiple organs in systemic sclerosis

Abstract

Systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of the skin and various internal organs. In SSc, telocytes, a peculiar type of stromal (interstitial) cells, display severe ultrastructural damages and are progressively lost from the clinically affected skin. The aim of the present work was to investigate the presence and distribution of telocytes in the internal organs of SSc patients. Archival paraffin-embedded samples of gastric wall, myocardium and lung from SSc patients and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were studied on tissue sections subjected to CD34 immunostaining. CD34/CD31 double immunofluorescence was performed to unequivocally differentiate telocytes (CD34-positive/CD31-negative) from vascular endothelial cells (CD34-positive/CD31-positive). Few telocytes entrapped in the fibrotic extracellular matrix were found in the muscularis mucosae and submucosa of SSc gastric wall. In the muscle layers and myenteric plexus, the network of telocytes was discontinuous or even completely absent around smooth muscle cells and ganglia. Telocytes were almost completely absent in fibrotic areas of SSc myocardium. In SSc fibrotic lung, few or no telocytes were observed in the thickened alveolar septa, around blood vessels and in the interstitial space surrounding terminal and respiratory bronchioles. In SSc, the loss of telocytes is not restricted to the skin, but it is a widespread process affecting multiple organs targeted by the fibrotic process. As telocytes are believed to be key players in the regulation of tissue/organ homoeostasis, our data suggest that telocyte loss might have important pathophysiological implications in SSc.

Keywords: CD34; fibrosis; gastric wall; immunohistochemistry; lung; myocardium; scleroderma; systemic sclerosis; telocytes.

Fig 1

Masson's trichrome-stained sections of gastric wall (A and B), LV myocardium (C and D) and lung (E and F) from controls and patients with systemic sclerosis (SSc). (B) Muscle layers of SSc gastric wall display accumulation of dense collagen bundles surrounding atrophic smooth muscle cells. (D) In SSc myocardium, cardiomyocytes are embedded in fibrous tissue. Inset: At higher magnification view, SSc cardiomyocytes display hypertrophic changes and diffuse cytoplasmic vacuolization. (F) Lung sections from SSc patients display the typical features of non-specific interstitial pneumonia with diffuse cellular inflammation and collagen deposition resulting in thickening of alveolar septa and severe distortion of the pulmonary parenchyma structure. All tissue sections are stained with Masson's trichrome with aniline blue (red colour: cytoplasm; blue colour: collagen). Scale bars are indicated in each panel.

Fig 2

Gastric wall specimens from controls (A, C and E) and patients with systemic sclerosis (SSc) (B, D and F). (A–F) CD34 immunoperoxidase labelling with haematoxylin counterstain. (A and B) Submucosa. (A) In control gastric wall submucosa, telocytes form a network surrounding vessels and ganglia (asterisk) and encircle the submucosal border of the muscularis propria. (B) In the submucosa of SSc gastric wall, very few telocytes entrapped in the fibrotic extracellular matrix are observed. Note the absence of telocytes around submucosal ganglia (asterisk). (C and D) Muscle layers. (C) In the muscularis propria of control gastric wall, numerous telocytes surround smooth muscle bundles and cells. Inset: At higher magnification view, telocytes display a slender nucleated body and very long varicose processes located in the interstitium between smooth muscle cells. (D) Telocytes are very few or absent in muscle layers of SSc gastric wall. (E and F) Myenteric plexus. (E) In control specimens, telocytes form a network enveloping myenteric plexus ganglia (arrows) and are present along the nerve strands of the interganglionic region. (F) Note the almost complete absence of telocytes around myenteric plexus ganglia (arrows) of SSc gastric wall. SM: submucosa; OM: oblique muscle layer; CM: circular muscle layer; LM: longitudinal muscle layer; MP: myenteric plexus. Scale bars are indicated in each panel.

Fig 3

Gastric wall specimens from controls (A–C) and patients with systemic sclerosis (SSc) (D–F). (A–F) Double immunofluorescence labelling for CD34 (green) and CD31 (red) with DAPI (blue) counterstain for nuclei. Telocytes are CD34-positive and CD31-negative, while vascular endothelial cells are CD34/CD31-double-positive. (A–C) Control gastric wall. Telocytes form a network around smooth muscle bundles and cells in the circular and longitudinal muscle layers. At the myenteric plexus, telocytes form a complex network enveloping the ganglia (arrow) and the nerve strands in the interganglionic region. Telocyte processes appear intermingled with ganglion cells. (D–F) SSc gastric wall. Telocytes are not present in the fibrotic areas of muscle layers. The network of telocytes is discontinuous or even almost completely absent around myenteric plexus ganglia (arrow) and nerve strands. CM: circular muscle layer; LM: longitudinal muscle layer; MP: myenteric plexus. Scale bars are indicated in each panel.

Fig 4

LV myocardium specimens from controls (A, B and E) and patients with systemic sclerosis (SSc) (C, D and F). (A–D) CD34 immunoperoxidase labelling with haematoxylin counterstain. (E and F) Double immunofluorescence labelling for CD34 (green) and CD31 (red) with DAPI (blue) counterstain for nuclei. (A and B) In control myocardium, numerous telocytes are located in the interstitium surrounding the cardiomyocytes. Inset: At higher magnification view, a myocardial telocyte displays a small fusiform cell body with two long processes placed between cardiomyocytes. (C and D) In the fibrotic areas of SSc myocardium, telocytes are almost completely undetectable. (E and F) Myocardial telocytes are CD34-positive and CD31-negative, while vascular endothelial cells are CD34/CD31-double-positive. Inset: Higher magnification view of a myocardial telocyte with three cellular extensions. Scale bars are indicated in each panel.

Fig 5

Lung specimens from controls (A–C and G) and patients with systemic sclerosis (SSc) (D–F and H). (A–F) CD34 immunoperoxidase labelling with haematoxylin counterstain. (G and H) Double immunofluorescence labelling for CD34 (green) and CD31 (red) with DAPI (blue) counterstain for nuclei. (A–C) In control lung parenchyma, telocytes are broadly distributed in the interstitial space around alveoli and bronchioles. Telocytes also encircle blood vessels (inset in A) and are located around smooth muscle bundles within the wall of bronchioles (inset in B). At higher magnification view, lung telocytes display a slender nucleated body and two long varicose processes extending in the pulmonary interstitium (inset in C). (D–F) In the fibrotic lung of SSc patients, very few or no telocytes are observed in the thickened alveolar septa and in the interstitial space surrounding terminal and respiratory bronchioles, around blood vessels (inset in D) and smooth muscle bundles within the wall of bronchioles (inset in E). (G and H) Pulmonary telocytes are CD34-positive and CD31-negative, while vascular endothelial cells are CD34/CD31-double-positive. Inset: Higher magnification view of a telocyte with two long cellular extensions. TB: terminal bronchiole. Scale bars are indicated in each panel.

Fig 6

Quantitative analysis of telocytes in sections of gastric wall (A, submucosa; B, muscle layers), myocardium (C) and pulmonary parenchyma (D) from controls and systemic sclerosis (SSc) patients. Data are represented as mean ± SD telocyte number per high-power field (hpf). *P < 0.05 versus control (by Student's t-test).