Mobilization of lymphatic endothelial progenitor cells and lymphatic neovascularization in primary Sjögren’s syndrome

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disorder charac- terized by chronic lymphocytic infiltration of exocrine glands leading to progressive functional impairment. Lympangiogenesis is a common finding in chronic inflamma- tory diseases; however, its role in pSS remains to be elucidated. Inflammation induces the production of growth factors for lymphatic vessels, such as vascular endothelial growth factor-C (VEGF-C). Recent growing evidence indicates that bone marrowderived lymphatic endothelial progenitor cells (LEPCs) may differentiate into lymphatic endothelial cells via VEGF-C/VEGFR-3 signaling contributing to lymphangiogenesis. This study was designed to investigate the circulating levels of LEPCs and the occurrence of lymphangiogenesis in pSS. Pheripheral blood mononuclear cells were collected from 10 female pSS patients and 11 healthy females. LEPCs, defined as CD34+CD133+VEGFR-3+ cells, were identified by FACS using CD34-FITC, CD133-APC and VEGFR-3-PE antibodies. Results were expressed as percentage of CD133+VEGFR-3+ cells among CD34+ cells. Labial minor salivary gland (MSG) biop- sies were obtained from 12 female pSS patients and 16 sicca non-pSS control females. MSGs were evaluated by haematoxylin-eosin and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germi- nal center-like structures. Lymphatic vessels were identified by immunohistochemistry for podoplanin (D2-40), a mucin-type transmembrane protein expressed by lymphatic endothelial cells but not by blood vessels. VEGF-C/VEGFR-3 expression in MSGs was investigated by immunofluorescence. An average ten-fold increase in circulating levels of LEPCs was found in pSS (35.2±2.7%) compared with controls (3.4±0.8%) (p=0.0003). In control MSGs, lymphatic vessels were only detected around excretory ducts in the interlobular connective tissue. In pSS MSGs, the number of lymphatic vessels was increased around interlobular excretory ducts and a newly formed lymphatic capil lary network was found within inflammatory foci. A strong expression of VEGF-C was detected in ductal cells, vessels and inflammatory cells in pSS MSGs. VEGFR-3 expres- sion was observed in a subset of vessels and infiltrating mononuclear cells. Our find ings suggest that LEPC mobilization and MSG lymphatic vessel reorganization may take center stage in the chronic inflammatory process of pSS

Morphological evidence of telocytes in human synovium

Abstract A new cell type named telocyte (i.e. cell with distinctive prolongations called telopodes) has recently been identified in the stroma of various organs in humans. However, no study has yet reported the existence of telocytes in the synovial membrane of diarthrodial joints. This work was therefore undertaken to search for telocytes in the normal human synovium using transmission electron microscopy, immunohistochemistry and immunofluorescence. Ultrastructural analyses demonstrated the presence of numerous spindle-shaped telocytes in the whole synovial sublining layer. Synovial telocytes exhibited very long and thin moniliform telopodes and were particularly concentrated at the boundary between the lining and sublining layers and around blood vessels. Light microscopy confirmed the presence of CD34-positive telocytes in the aforementioned locations. Moreover, synovial telocytes coexpressed CD34 and platelet-derived growth factor receptor α. Double immunostaining further allowed to unequivocally differentiate synovial telocytes (CD34-positive/CD31-negative) from vascular endothelial cells (CD34-positive/CD31-positive). The in vitro examination of fibroblast-like synoviocyte primary cultures revealed the coexistence of different cell types, including CD34-positive telocytes projecting typical moniliform telopodes. In conclusion, our work provides the first evidence that telocytes do exist in the human synovium and lays the groundwork for future studies on synovial telocytes in a variety of degenerative and destructive joint diseases

Telocytes constitute a widespread interstitial meshwork in the lamina propria and underlying striated muscle of human tongue

Abstract Telocytes have recently emerged as unique interstitial cells defined by their extremely long, thin and moniliform prolongations termed telopodes. Despite growing evidence that these cells consistently reside in the stromal compartment of various organs from human beings, studies dealing with telocytes in structures of the oral cavity are scarce. Hence, the present morphologic study was undertaken to explore for the first time the presence and specific localization of telocytes within tissues of the normal human tongue, a complex muscular organ whose main functions include taste, speech, and food manipulation in the oral cavity. Telocytes were initially identified by CD34 immunostaining and confirmed by CD34/PDGFRα double immunofluorescence and transmission electron microscopy. CD34+/PDGFRα+ telocytes were organized in interstitial meshworks either in the tongue lamina propria or in the underlying striated muscle. Lingual telocytes were immunonegative for CD31, c-kit and α-SMA. Telopodes were finely distributed throughout the stromal space and concentrated beneath the lingual epithelium and around CD31+ vessels, skeletal muscle bundles/fibers, and intramuscular nerves and ganglia. They also enveloped salivary gland units outside the α-SMA+ myoepithelial cells and delimited lymphoid aggregates. These findings establish telocytes as a previously overlooked interstitial cell population worth investigating further in the setting of human tongue pathophysiology

A loss of telocytes accompanies fibrotic remodelling of the colonic wall in ulcerative colitis

Crohn’s disease (CD) and ulcerative colitis (UC) are complex diseases in which the interaction of genetic, environmental and microbial factors drives chronic relapsing and remitting intestinal inflammation that finally leads to extensive tissue fibrosis. In UC, this results in a stiff, fibrotic colon unable to carry out peristalsis or to resorb fluids. Colonic dysmotility is often observed in UC patients and has been linked to severe damages of the enteric neural structures and a reduced density of interstitial cells of Cajal (ICC). Telocytes (TC), a peculiar type of stromal cells, have been recently identified in a variety of human tissues and organs, including the gastrointestinal tract. Several roles have been proposed for TC, including mechanical support, spatial relationships with different cell types, intercellular signalling and modulation of intestinal motility by spreading the slow waves generated by the pacemaker ICC. We have recently demonstrated that a loss of TC accompanies the fibrotic remodelling of the intestinal wall in CD patients. The aim of the present work was to investigate the presence and distribution of TC in colonic specimens from UC patients compared with controls. Archival paraffin-embedded full-thickness 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. TC were identified by CD34 immunohistochemistry. Double immunofluorescence for CD34 and CD31 (vascular endothelial cells), alpha-SMA (smooth muscle cells, myofibroblasts) and c-kit (ICC) was also performed. 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 TC were found in the muscularis mucosae and submucosa of both early and advanced fibrotic UC colonic wall. Conversely, numerous myofibroblasts were observed in the submucosa of all UC cases. In the muscle layers and at the myenteric plexus of early fibrotic UC, TC were preserved in their distribution. In the muscularis propria of advanced fibrotic UC, the network of TC was reduced or even completely absent around smooth muscle cells and myenteric plexus ganglia, paralleling the loss of the ICC network. In UC, the loss of TC accompanies the fibrotic remodelling of the colonic wall and might contribute to colonic dysmotility

Scleroderma-like Impairment in the Network of Telocytes/CD34+ Stromal Cells in the Experimental Mouse Model of Bleomycin-Induced Dermal Fibrosis

Considerable evidence accumulated over the past decade supports that telocytes (TCs)/CD34+ stromal cells represent an exclusive type of interstitial cells identifiable by transmission electron microscopy (TEM) or immunohistochemistry in various organs of the human body, including the skin. By means of their characteristic cellular extensions (telopodes), dermal TCs are arranged in networks intermingled with a multitude of neighboring cells and, hence, they are thought to contribute to skin homeostasis through both intercellular contacts and releasing extracellular vesicles. In this context, fibrotic skin lesions from patients with systemic sclerosis (SSc, scleroderma) appear to be characterized by a disruption of the dermal network of TCs, which has been ascribed to either cell degenerative processes or possible transformation into profibrotic myofibroblasts. In the present study, we utilized the well-established mouse model of bleomycin-induced scleroderma to gain further insights into the TC alterations found in cutaneous fibrosis. CD34 immunofluorescence revealed a severe impairment in the dermal network of TCs/CD34+ stromal cells in bleomycin-treated mice. CD31/CD34 double immunofluorescence confirmed that CD31−/CD34+ TC counts were greatly reduced in the skin of bleomycin-treated mice compared with control mice. Ultrastructural signs of TC injury were detected in the skin of bleomycin-treated mice by TEM. The analyses of skin samples from mice treated with bleomycin for different times by either TEM or double immunostaining and immunoblotting for the CD34/α-SMA antigens collectively suggested that, although a few TCs may transition to α-SMA+ myofibroblasts in the early disease stage, most of these cells rather undergo degeneration, and then are lost. Taken together, our data demonstrate that TC changes in the skin of bleomycin-treated mice mimic very closely those observed in human SSc skin, which makes this experimental model a suitable tool to (i) unravel the pathological mechanisms underlying TC damage and (ii) clarify the possible contribution of the TC loss to the development/progression of dermal fibrosis. In perspective, these findings may have important implications in the field of skin regenerative medicine

Experimental model of rat ileitis resembling Crohn’s disease

Crohn’s disease (CD) is a chronic, idiopathic and relapsing inflammatory disease of the gastrointestinal (GI) tract. Current theories point to an impaired immune response to microbes within the intestinal flora in a genetically susceptible host. Although the lymphatic obstruction represents one of the main histopathological diagnostic criteria, its etiology is unknown. Many animal models that, in some respects, resemble human CD have been developed, but these models do not represent the complexity of human disease. We planned to obtain an animal model of CD by surgical manipulation of the enteric lymphatic vessels in the TNBS model of rat ileitis. Surgical approach: 12 F344 male rats were surgically treated by ligature and cut of ileo-colic lymphatic chain near the Treitz. The lymph nodes near the ileocecal valve were injected with a sclerosing agent (control group). Surgical and chemical approach: F344 were treated as above and, at the same time, intraluminally ileum injected with TNBS, a chemical reagent able to induce severe enteritis. 6 rats were sacrificed after one week (group 1). 8 rats received two more weekly TNBS injections and then were sacrificed (group 2). Control and treated rats were sacrificed at the same time. Animals were weighted and monitored for clinical manifestations of GI inflammation. At sacrifice, macroscopical changes were noted and ileum and colon specimens were harvested and processed for histopathological analysis. Most of the animals showed adhesions of bowel loops and thickening of the mesentery. Groups 1 and 2: TNBS induced more severe macroscopical features of inflammation in the intestinal wall, such as fistolae, dilatation of the ileum and sometimes intestinal obstruction. Microscopically, we observed transmural inflammatory infiltrate, fibrosis of mesentery, granulomas, and in the most severe cases, signs of neoangiogenesis, derangement of the muscle layers, ulcers and loss of villi in the mucosa. Besides the microscopical inflammatory changes, we evidenced an increased number of dilated lymphatic vessels. Group 1: one week after a single injection, most of the animals partially solved and showed less severe microscopical damages. The surgical and chemical approach did not induce in this rat model the severe fibrosis and the stricture of intestinal lumen that usually lead to surgery in human CD. However, the histopathological features found in these rats resemble the severe pathological features observed in human CD

Altered Immunoregulation in Rheumatoid Arthritis: The Role of Regulatory T Cells and Proinflammatory Th17 Cells and Therapeutic Implications

In recent years several studies investigated the role of T lymphocyte subpopulations in the pathogenesis of rheumatoid arthritis (RA). Pathogenic Th17 cells mediate pannus growth, osteoclastogenesis, and synovial neoangiogenesis; hence they are key players in the development of the disease. On the other hand, regulatory T (Treg) cells are a T cell subset whose peculiar function is to suppress autoreactive lymphocytes. The imbalance between Th17 and Treg cells has been identified as a crucial event in the pathogenesis of RA. In addition, the effects of currently employed RA therapeutic strategies on these lymphocyte subpopulations have been extensively investigated. This review article aims to discuss current knowledge on Treg and Th17 cells in RA and possible implications of their therapeutic targeting in this disorder

A two-step immunomagnetic microbead-based method for the isolation of human primary skin telocytes/CD34+ stromal cells

Telocytes (TCs), commonly referred to as TCs/CD34+ stromal cells, are a peculiar type of interstitial cells with distinctive morphologic traits that are supposed to exert several biological functions, including tissue homeostasis regulation, cell-to-cell signaling, immune surveillance, and reparative/regenerative effects. At present, the majority of studies investigating these cells are mainly descriptive and focus only on their morphology, with a consequent paucity of functional data. To gain relevant insight into the possible functions of TCs, in vitro analyses are clearly required, but currently, the protocols for TC isolation are only at the early stages and not fully standardized. In the present in vitro study, we describe a novel methodology for the purification of human primary skin TCs through a two-step immunomagnetic microbead-based cell separation (i.e., negative selection for CD31 followed by positive selection for CD34) capable of discriminating these cells from other connective tissue-resident cells on the basis of their different immunophenotypic features. Our experiments clearly demonstrated that the proposed method allows a selective purification of cells exhibiting the peculiar TC morphology. Isolated TCs displayed very long cytoplasmic extensions with a moniliform silhouette (telopodes) and presented an immunophenotypic profile (CD31−/CD34+/PDGFRα+/vimentin+) that unequivocally differentiates them from endothelial cells (CD31+/CD34+/PDGFRα−/vimentin+) and fibroblasts (CD31−/CD34−/PDGFRα+/vimentin+). This novel methodology for the isolation of TCs lays the groundwork for further research aimed at elucidating their functional properties and possible translational applications, especially in the field of regenerative medicine