Figure 3.

PBEF exerts its antiapoptotic activity as a secreted factor. (A) Naive neutrophils were incubated for 21 hours with conditioned medium from resting or LPS-stimulated neutrophils, with or without prior transfection with PBEF antisense oligonucleotide or the sense control. Conditioned medium from resting neutrophils did not alter apoptotic rates in comparison with untreated controls (white bar). Conditioned medium from neutrophils incubated with LPS inhibited the apoptosis of naive neutrophils (*P < 0.05 versus control cell supernatants). Pretreatment with an antisense PBEF oligonucleotide, but not with the sense control, blocked this inhibitory activity (^#P < 0.05 versus sense control). Data are means ± SD of n = 6 experiments. (B) Culture supernatants or whole cell lysates from CHO cells transfected with a pCDNA3.1 vector carrying a PBEF/c-myc construct were immunoprecipitated with anti_c-myc. PBEF could be detected by Western blot analysis with anti-PBEF Ab in both lysates and supernatants from transfected cells, but not in those from nontransfected cells or cells transfected with plasmid containing c-myc alone. (C) Supernatants from transfected CHO cells suppressed the apoptosis of resting neutrophils as measured by propidium iodide uptake, whereas supernatants from vector-treated controls were without effect; *P < 0.05 versus control or empty vector, n = 4. (D) A recombinant PBEF/GST fusion protein added to cultures of control neutrophils induced dose-dependent inhibition of apoptosis; polymyxin B (10 ∝g/ml) was added to cultures to neutralize any contaminating LPS. Recombinant GST alone was without effect (data not shown). Apoptosis was measured by propidium iodide uptake at 21 hours; results are means ± SD of n = 3 studies.