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Activated T lymphocytes induce apoptosis of osteoblastic cells derived from stromal cell line st2

Background/aims: The immune system regulates the number and differentiation of bone cells via different cellular and soluble factors. Among factors expressed by activated T lymphocytes, Fas ligand (FasL) may induce bone loss by inhibiting osteoblast and stimulating osteoclast function. The aim of our study was to investigate the ability of FasL expressed by activated T lymphocytes to induce apoptosis of osteoblasts derived from murine ST2 stromal cell line. Methods: Osteoblast differentiation of ST2 cells was induced by ascorbic acid and analyzed by alkaline phosphatase (AP) activity, AP histochemistry, and qPCR analysis of osteoblast specific gene expression (Runx2, AP, osteocalcin, osteoprotegerin). Fas expression and apoptosis of ST2 cells were determined by flow cytometry. Lymph node T lymphocytes from C57Bl/6 mice were activated with concanavalin A (ConA) and added to ST2 cultures on days 7 and 10. T lymphocyte activation was confirmed by flow cytometry as an increase in CD69 expression. Expression of FasL in activated T lymphocytes was analyzed by qPCR. Results: Ascorbic acid induced osteoblastic differentiation of ST2 cells was confirmed by significant increase in AP activity, proportion of AP positive cells, and expression of osteoblast specific genes. According to the flow cytometry analysis, 95% of ST2 cells expressed Fas. ConA stimulation induced a 6-fold increase in FasL gene expression in T lymphocytes. Annexin/PI staining demonstrated that the addition of activated T lymphocytes increased the percentage of apoptotic ST2 cells compared with untreated ST2 cell cultures on day 14 (40% vs. 14 %), as well as the percentage of dead cells (8% vs. 2%). Conclusion: Activated T lymphocytes have the ability to induce apoptosis of ST2 cells, which is at least partially caused by binding of FasL expressed on activated T lymphocytes to constitutively expressed Fas on ST2 cells. Activated T lymphocytes may be further used as a physiological model for studying Fas mediated osteoblast apoptosis and inhibition of differentiation. Analysis of signaling molecules involved in this interaction may define new therapeutic targets for bone loss in inflammatory diseases.

apoptosis; CD98; osteoblast; stromal cells; T lymphocytes

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