LPS-induced apoptosis in macrophages from TNF-R KO mice.

(A) LPS, but not TNF-α, induces apoptosis in macrophages from TNF-αR KO mice. Macrophages (10⁵) from either wild-type or TNF-αR KO mice were treated for 24 hours with LPS (100 ng/mL) or TNF-α (100 ng/mL). DNA fragmentation was evaluated by measuring histone-associated DNA fragments by ELISA. (B) Time course of LPS-induced apoptosis in macrophages from TNF-αR KO mice. Cells from control and KO mice were treated with LPS (100 ng/mL) for the indicated periods of time. Apoptosis was determined as indicated previously. (C) Macrophages from TNF-αR KO mice express iNOS in response to LPS. Macrophages were treated with 100 ng/mL of LPS for the indicated times; 20 μg of total RNA per lane was analyzed by Northern blotting. (D) Production of NO in macrophages from TNF-αR KO mice. The production of NO was measured in cultures of macrophages from each group of mice stimulated with 100 ng/mL of LPS for the indicated times in the presence or absence of 20 μmol/L SMT. (E) SMT blocks LPS-induced apoptosis in macrophages from TNF-αR KO mice but not in control macrophages. Macrophages (10⁵) from control and KO mice were treated with LPS (100 ng/mL) for the indicated periods of time in the presence or absence of SMT (20 μmol/L). Apoptosis was determined by ELISA. Each experiment was performed in triplicate and represented as the mean ± SD. (F) LPS-induced apoptosis in macrophages from TNF-αR KO mice is mediated by NO production. DNA fragmentation was analyzed in a 2% agarose gel electrophoresis. Macrophages of each group were treated with 100 ng/mL of LPS in the presence or absence of either 20 μmol/L SMT (iNOS inhibitor), 50 μmol/L SNAP (NO donor), or 100 ng/mL rmTNF-α.