Table 2.

Intracellular levels of active caspase-3 and -8 do not differ in eosinophils undergoing apoptosis induced by activating the extrinsic apoptosis pathway in vitro

Time point	WT EosinophilsA	Adam8−/− Eosinophils	WT Eosinophils	Adam8−/− Eosinophils
	Active caspase-3 in ng/ml Mean (SEM) n	Active caspase-3 in ng/ml Mean (SEM) n	Active caspase 8 in units/ml Mean (SEM) n	Active caspase 8 in units/ml Mean (SEM) n
0 h	6.4 (8) 4B	13.4 (14.6) 4	0.1 (0.4) 3	0.7 (0.4) 3
2 h	25.1 (10.2) 4	16.1 (13.8) 4	0.5 (0.6) 3	0.6 (0.5) 3
4 h	33.9 (10.6) 4	27.8 (13.3) 4	1.8 (1.4) 3	1.5 (0.9) 3
6 h	63.7 (15.9) 4	44.8 (18.3) 4	2.5 (1.9) 3	1.2 (1.2) 3
8 h	57.5 (18.1) 4	49.1 (23.1) 4	2.1 (1.9) 3	0.6 (0.9) 3
20 h	12.8 (5.9) 4	6.6 (2.9) 4	0 (0.5) 3	0 (0.6) 3

^AEosinophils were isolated from the bone marrow of unchallenged BALB/c WT and BALB/c Adam8^−/− mice, as described in Methods. Macrophages were isolated from the peritoneal cavities of BALB/c WT and BALB/c Adam8^−/− mice 4 days after thioglycollate was delivered by the i.p. route, and cells were cultured for 5 days in tissue culture plates to render them quiescent. Equal numbers of eosinophils or macrophages were incubated with a cross-linking antibody to CD95 (FAS) which activates this death domain-containing receptor, or a non-immune isotype control antibody for up to 20 h for eosinophils or 48 h for macrophages. At intervals, cell extracts were prepared at 5 × 10⁶ cells/ml and intracellular levels of active caspase-3 and -8 were measured using specific fluorogenic substrates, as described in Methods.

^BData are mean (SEM); n =3–4 mice studied per group.

We were not able to induce significant activation caspase-3 or 8 in either WT or Adam8^−/− macrophages by treating cells with the FAS cross-linking antibody for up to 48 h (data not shown).