Purpose: To study the bystander effects of gamma-radiation in murine lymphocytes using irradiated conditioned medium (ICM) generated from irradiated lymphocytes.
Methods: Proliferation response of unirradiated lymphocytes to mitogen concanavalin A (con A) in presence of ICM, collected from gamma-irradiated lymphocytes (60Co source; 0.35 Gy/min; 0.1-1 Gy), was studied by 3H-thymidine incorporation and also by dye dilution using carboxyfluorescein succinimidyl ester (CFSE). Expression of proliferation markers, interleukin 2 receptor alpha chain (CD25) and cyclin D in ICM treated lymphocytes was analyzed by labeling with specific antibodies. Intracellular reactive oxygen species (ROS) and apoptosis were estimated by flow cytometry using dichlorodihydrofluorescein diacetate (H2DCFDA) and propidium iodide, respectively. Nitric oxide (NO) was measured using Griess reagent.
Results: Proliferation response to con A in unirradiated lymphocytes was enhanced in the presence of ICM with maximum enhancement observed in the presence of 0.5 Gy ICM. Augmentation of proliferation in the presence of ICM was accompanied by an increase in CD25 and cyclin D expression, enhanced ROS and NO generation. ICM pretreated lymphocytes showed adaptive response to radiation which was not abrogated by wortmannin, a phosphatidyl inositol 3-kinase (PI3K) inhibitor.
Conclusion: Soluble factors released from irradiated lymphocytes initiate a signaling cascade in unirradiated lymphocytes resulting in increased response to mitogen and radioresistance which may have an important role in radiation-induced immunomodulation.