Microvesicles contain both immature and mature IL-1β. In panel A, immature (Imm) and mature (Mat) DCs were seeded in culture flasks at a concentration of 106/mL and treated with 200 μM BzATP at 37°C for 10 minutes in sucrose solution. Interleukin-1β was measured in vesicle-free DC supernatant (▪), intact microvesicles (⊡), and microvesicles lysed by 3 freeze-thawing cycles (□). Data are averages ± SE of 3 experiments from 3 different donors each performed in triplicate. Interleukin-1β content was also assessed by Western blot in microvesicles from immature and mature DCs treated with BzATP in sucrose or in standard saline (B); in panel D, secretion of the cytokine was analyzed in the supernatant (s) of mature DCs treated with BzATP in standard saline; microvesicle content of IL-1β is shown for comparison (ves). Release of cathepsin D from mature DCs has been assessed in supernatants and vesicles (C and E, respectively) in the absence or presence of 20 μM BEL (c indicates control). Panel F shows the effect of BEL on microvesicle IL-1β content. Cells were incubated in the presence of BEL in RPMI 1640 medium for 1 hour at 37°C.
