The effects of T cells and their products on in vitro healing of epitenon cell microwounds.

The purpose of the present investigation was to study the activity and behaviour of rat epitenon cells cultured in the presence of activated helper/inducer T lymphocytes and T-cell-derived cytokines interleukin-1 (IL-1), IL-2, transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma). We measured the speed of healing of microwounded monolayers of epitenon cells as well as intercellular adhesion, cell proliferation and fibronectin production. The speed of monolayer healing was increased in the presence of activated CD4+ T lymphocytes and cytokines: TGF-beta > IL-2 > IL-1 > IFN-gamma. TGF-beta and IL-2 were also found to promote cell-cell adhesion; other cytokines had a minor effect. IL-2, IL-1 and IFN-gamma promoted [3H]thymidine incorporation in epitenon cells whereas TGF-beta had an inhibitory effect. Fibronectin production was studied with immunofluorescence staining methods. Activated CD4+ T lymphocytes and TGF-beta stimulated the deposition of fibronectin whereas other cytokines did not have a significant effect. Our results suggest that activated T lymphocytes and T-cell-derived cytokines, especially IL-2 and TGF-beta play a crucial role in the regulation of epitenon cell proliferation, adhesion and extracellular matrix production during in vitro microwound healing. As epitenon cells are the main cell type participating in tendon repair, factors regulating their activity may find application in clinical practice.

An improved method for the isolation of cells from tissues: prevention of cell rounding enhances spreading and locomotion of keratinocytes (epitheliocytes).

Benzamide (10mM) in Ca-free solution anesthetizes small cold blooded animals such as tadpoles of Rana temporaria, Xenopus laevis and an aquarium fish, guppy (Lebistes [Poecilia] reticulatus). Trypsinization of tails of tadpoles in Ca-free, benzamide-supplemented solution permits isolation of keratinocytes (epitheliocytes) maintaining polygonal shapes and prevents cell rounding. Such cells quickly attach to glass, spread and commence locomotion in contrast to those cells which assume spherical shape after standard trypsinization.

The accumulation of inflammatory cells in synovial sheath and epitenon during adhesion formation in healing rat flexor tendons.

The accumulation of inflammatory cells in synovial tissue was studied using indirect immunofluorescence assays on cell cultures and frozen tissue sections of healing rat digital flexor tendons. Flexor tendons were collected from rats 3, 7 and 14 days after crush injury. Tendon sheath and epithenon cells were isolated by sequential enzymic digestion and cultured for 2 days. Subpopulations of synovial and inflammatory cells were identified with MoAbs against cell surface glycoproteins present on B lymphocytes (CD45), T lymphocytes (CD2, CD4, CD8), macrophages (CD14) and endothelial cells. A phagocytosis assay was also used to identify macrophages. We report a substantial increase in the number of T lymphocytes (mainly helper/inducer) and phagocytotic cells with monocyte/macrophage surface markers in tendon sheath and epitenon 3 days after crush injury. The infiltration of inflammatory cells into synovial sheath and epitenon preceded an increase in fibronectin production by tendon cells which was seen 7 days after injury. To study the interaction between T lymphocytes and synovial cells in vitro, we established synovial fibroblast-like type B cell cultures and used stimulated and non-stimulated T lymphocytes in cell binding assays. We observed increased adhesiveness between unstimulated synovial cells and synovial cells previously cultured with activated and non-activated T lymphocytes. ELISA inhibition studies have shown an increase in fibronectin production by synovial fibroblasts co-cultured with stimulated CD4+ T lymphocytes. We suggest that the presence of inflammatory cells in synovial sheath and epitenon during tendon healing induces synovial fibroblasts and epitenon cells to increase their production of fibronectin, which provides a scaffold for subsequent adhesion formation.

T-cell-mediated response in Dupuytren's disease.

The cause of Dupuytren's disease is unknown, but inflammatory cells might have a role. Enzymatic digestion of diseased tissue permits identification and immunofluorescent labelling of a cell subset displaying inflammatory cell morphology. Cytofluorimetry of this cell population demonstrated the presence of CD3-positive lymphocytes and expression of major histocompatibility complex (MHC) class II proteins. These results raise the possibility that Dupuytren's disease is a T-cell-mediated autoimmune disorder. The development of medical treatment on this basis may reduce the need for surgery, with its associated morbidity and high recurrence rates.

Ehrlich ascites tumour cell spreading is induced upon contact with immobilized isolated fibroblast plasma membranes and fibronectin.

Ehrlich ascites tumour (EAT) cells normally growing in suspension, adhere and spread upon contact with fibroblasts. Changes in EAT cell morphology and adhesiveness induced in contact with purified fibroblast plasma membranes, fibroblast extracellular matrix and fibronectin were investigated. EAT cells adhered readily to glass covered with fibroblast extracellular matrix or fibronectin, but they did not spread. Immobilization of fibroblast plasma membranes or fibronectin on nitrocellulose restored EAT cell ability to spread. Tumour cells spread on immobilized plasma membranes or fibronectin maintained a "fried egg" morphology, while these spread on living fibroblasts were rather elongated. EAT cells preincubated with anti-fibronectin serum lost their ability to adhere to any of the tested substrata: living and fixed fibroblasts, fibroblast extracellular matrix, nitrocellulose with immobilized fibroblast plasma membranes or fibronectin. When anti-fibronectin serum was applied only to these substrata and it was washed out before EAT cell plating, EAT cell adhesion and spreading were not significantly changed. Extraction of extracellular matrix components from immobilized fibroblast plasma membranes did not influence EAT adhesion and spreading. The results are discussed with reference to the specific role of fibronectin in the growth of EAT cells as a solid tumour or suspension.