Sphingosine-1-phosphate conveys protection. (A-C) Apoptosis in human primary macrophages was induced for 8 hours with 10 ng/mL TNF-α and 10 μg/mL CHX. (A) Cells remained untreated or were treated with ACM, the ACM-lipid fraction, the ACM-aqueous fraction, ACM derived from Jurkat cells exposed to DMS prior to initiation of apoptosis (ACM-DMS), or the ACM-lipid fraction specific for S1P extraction (S1P-extraction), 16 hours prior to induction of cell death. Caspase-3 activity was quantified as described in “Materials and methods.” Data are presented as the mean ± SD from 4 independent experiments. Significant differences between samples are marked with an asterisk (P < .01). (B) Cells were not treated, exposed to the ACM, or stimulated with 10 μM S1P for 16 hours. Western analysis shows expression of Bcl-2, Bcl-X_L, and phosphorylated Ser136-Bad. One representative experiment of 3 is displayed. Histograms show quantification of Western data normalized to actin by using AIDA software. Data are presented as the mean ± SEM from 3 independent experiments. (C) Cells remained untreated or were exposed for 16 hours to ACM alone or to ACM after pretreatment with 100 nM FTY720 for 1 hour followed by further incubations of 48 hours. Afterward, TNF-α/CHX treatments lasted for 8 hours. Data are presented as the mean ± SD from 4 independent experiments. Significant differences between samples are marked with an asterisk (P < .05).