Decreases in CD31 and CD47 Levels on the Cell Surface during Etoposide-Induced Jurkat Cell Apoptosis

Engulfment of apoptotic cells is regulated by 'eat me' and 'don't eat me' signals on the cell surface. Alterations to the 'eat me' signals have been well described; however, very little is known about the 'don't eat me' signals on the cell surface during apoptosis. In the present study, apoptosis of Jurkat cells was induced by treatment with topoisomerase II inhibitor etoposide, and then the CD31 and CD47 levels on the apoptotic cell surface and in microparticles were estimated by flow cytometry and immunoblotting methods in the presence of caspase, metalloproteinase, and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitors. The CD31 and CD47 levels on the cell surface of apoptotic Jurkat cells had decreased after treatment with etoposide. These decreases in CD31 and CD47 levels on the apoptotic cell surface were almost completely suppressed by the caspase 3 inhibitor, Ac-DEVD-CHO, and partially suppressed by caspase 8 (Ac-IETD-CHO) and caspase 9 (Ac-LEHE-CHO) inhibitors but not by the metalloproteinase inhibitors GM6001 and TAPI-0. Microparticle counts in culture supernatants were higher during etoposide-induced apoptosis. The ROCK1 inhibitor, Y27632, suppressed blebbing formation and microparticle release. Moreover, flow cytometry and immunoblotting revealed CD31 and CD47 in the microparticles. These results indicate that CD31 and CD47 were released by the apoptotic Jurkat cells into the culture supernatant in microparticles, but not in soluble forms, resulting in decreased levels on the apoptotic cell surface.