Secretion of both cathepsin D and IL-1β by DCs is dependent on [Ca⁺⁺]_i increases.

(A) LPS-activated DCs cultured 6 hours without (lanes 1, 2, 5, and 6) or with (lanes 3, 4, 7, and 8) alloreactive T cells at a DC/T cell ratio of 1:10 were untreated (−, lanes 1, 3, 5, and 7) or stimulated for the last 10 minutes with 1 mM ionomycin (+, lanes 2, 4, 6, and 8). Supernatants were TCA concentrated and analyzed by Western blot for their content in IL-1β (lanes 1-4) and cathepsin D (lanes 5-8). Panels B and C show the densitometric analyses of IL-1β, procathepsin D, and cathepsin D present in supernatants of LPS-activated DCs, cultured 6 hours alone (B, DCs) or with alloreactive T cells (C, DCs + allo T) in the presence or absence of different substances for the times indicated: 5 mM EGTA (during the entire time of incubation), 10 μM nifedipine (NFD, 30 minutes of treatment, before coculture with T cells), 1 mM ionomycin (iono, for the last 10 minutes of incubation), 10 μM Bay K8644 (BayK, for the last 15 minutes of incubation), 10 nM thapsigargin (thapsi, for the last 30 minutes of incubation), or 10 nM thapsigargin and 5 mM EGTA (thapsi + EGTA, for the last 30 minutes of incubation). As a control, DCs were fixed for 10 minutes with 1% glutaraldehyde (C, GA-treated DCs + allo T) and secretion induced by T cells was evaluated with or without ionomycin stimulation. One representative experiment of 3 is shown.