Ionizing radiation (Ir) Alters functional activity of cell surface fas and fas ligand (Fasl) In human bone marrow stromal cells

Hematopoietic homeostasis requires a network of cytokines that balances proliferation and differentiation with apoptosis of stem/progenitor cells. We have reported that Fas and FasL, principal mediators of apoptosis, are modulated at the cell surface by IR (Albanese and Dainiak, Radiat Res 153:49, 2000). Here, we examined expression and functional activity of Fas and FasL in a cytokine-producing, human bone marrow stromal cell line (HS-5), before and after exposure to IR. Plasma membranes were purified from HS-5 cells, solubilized in Triton-X 100 and subjected to Western blot analysis. Proteins of 40 and 48 kDal, corresponding to FasL and Fas, respectively, were observed, a finding confirmed by flow cytometry and RT-PCR. Despite the presence of Fas receptor, anti-Fas antibodies DX-2 and CH.11 (100–500 ng/ml), failed to induced cell death in HS-5 cells, as determined by the MTT assay. Hs-5 cells exposed to IR (0–10 Gy), displayed a dose-dependent increase in levels of surface Fas and FasL, as assessed by flow cytometry. To examine the biological activity of FasL expressed by HS-5 cells, irradiated and non-irradiated HS-5 cells (effectors) were co-cultured with Jurkat cells (targets), a cell line sensitive to FasL. Viability of target cells was determined using the MTT assay following a 24 hr co-incubation period. We observed that Jurkat cell viability increased in a dose-dependent manner in samples co-cultivated with HS-5 cells exposed to 0–10 Gy of IR. A maximum increase (relative to non-irradiated HS-5 cells) in target cell survival was observed in Jurkat cell samples co-cultered with HS-5 cells that were previously irradiated (10 Gy). These findings suggest that HS-5 cells express FasL at their surface in a biologically active form and that functional activity of FasL is diminished following exposure to IR. Whether the reduction in apoptosis-inducing activity of FasL results from direct damage by IR to the protein or to the proteinʹs lipid microenvironment is unclear. The finding that Fas is expressed by HS-5 cells in an inactive form may explain why these cells fail to undergo apoptosis when grown in monolayer.