1. LPS induces apoptosis in macrophages mostly through the autocrine production of TNF-α
by Jordi Xaus, Mònica Comalada, Annabel F. Valledor, Jorge Lloberas, Francisco López-Soriano, Josep M. Argilés, Christian Bogdan, and Antonio Celada
Blood
Volume 95(12):
June 15, 2000
©2000 by American Society of Hematology

2. LPS induces apoptosis in bone marrow macrophages
LPS induces apoptosis in bone marrow macrophages.(A and B) 106 macrophages were stimulated with 100 ng/mL of LPS for 6 hours.
LPS induces apoptosis in bone marrow macrophages.(A and B) 106 macrophages were stimulated with 100 ng/mL of LPS for 6 hours. DNA was stained with DAPI, and induction of apoptosis was analyzed by cytometric analysis (A) or visualizing the cells in a fluorescence microscope (B). Apoptotic cells are marked with arrows. (C and D) LPS induces apoptosis in a time-dependent manner. Apoptotic DNA from macrophages treated with LPS (100 ng/mL) for the indicated times was analyzed by agarose gel electrophoresis (C) or by using an ELISA technique (D). (E) LPS induces apoptosis in a dose-dependent fashion; 105 macrophages were treated for 12 hours with the indicated concentrations of LPS. Apoptotic DNA was measured as in (D). (F) Apoptosis induced by LPS depends on the presence of FBS. The cells were treated with 100 ng/mL LPS for 12 hours in the presence of the indicated concentrations of FBS. Fragmentation of DNA was measured by ELISA. The ELISA experiments were performed in triplicate and represented as the mean value ± SD. These figures are representative of 4 independent experiments.
Jordi Xaus et al. Blood 2000;95:
©2000 by American Society of Hematology

3. Exogenous NO induces apoptosis in bone marrow macrophages
Exogenous NO induces apoptosis in bone marrow macrophages.(A) NO production by SNAP; 105 macrophages were treated with 50 μmol/L of SNAP for the indicated times and the production of NO was determined.
Exogenous NO induces apoptosis in bone marrow macrophages.(A) NO production by SNAP; 105 macrophages were treated with 50 μmol/L of SNAP for the indicated times and the production of NO was determined. (B) Time course of SNAP-induced apoptosis; 105 macrophages were treated with 50 μmol/L SNAP at the indicated times. Apoptosis was measured by ELISA. (C) The cells were treated for 24 hours with the indicated concentrations of SNAP, and apoptosis was detected as indicated above. Each experiment was performed in triplicate and the results of 1 representative of 2 independent experiments are represented as the mean value ± SD.
Jordi Xaus et al. Blood 2000;95:
©2000 by American Society of Hematology

4. LPS induces the expression of iNOS and the production of NO
LPS induces the expression of iNOS and the production of NO.The expression of iNOS induced by LPS was measured by Northern blot (A) or Western blot (B) as described in the “Materials and methods” section.
LPS induces the expression of iNOS and the production of NO.The expression of iNOS induced by LPS was measured by Northern blot (A) or Western blot (B) as described in the “Materials and methods” section. (C) LPS induces the production of NO; 106 BMDM cultured in media without phenol-red were stimulated with 100 ng/mL LPS, and the supernatants were harvested at the indicated times. NO production was measured as the nitrite/nitrate levels. Each point was performed in triplicate and represented as the mean ± SD. These figures are representative of 3 independent experiments.
Jordi Xaus et al. Blood 2000;95:
©2000 by American Society of Hematology

5. Treatment of macrophages with SMT inhibits LPS-induced NO production but not apoptosis.(A) The expression of iNOS was measured by Northern blot in macrophages treated with 100 ng/mL of LPS in the presence or absence of SMT, an iNOS inhibitor (20 μmol/L).
Treatment of macrophages with SMT inhibits LPS-induced NO production but not apoptosis.(A) The expression of iNOS was measured by Northern blot in macrophages treated with 100 ng/mL of LPS in the presence or absence of SMT, an iNOS inhibitor (20 μmol/L). (B) SMT inhibits LPS-induced NO production; 106 macrophages were treated with 100 ng/mL of LPS for the indicated times in the presence or absence of SMT (20 μmol/L). The production of NO was assessed by determination of the nitrate/nitrite levels. (C) SMT did not inhibit LPS-induced apoptosis. The cells were treated with 100 ng/mL of LPS for the indicated times in the presence or absence of SMT (20 μmol/L). Each experiment was performed in triplicate, and the results of 1 representative of 2 independent experiments are represented as the mean value ± SD.
Jordi Xaus et al. Blood 2000;95:
©2000 by American Society of Hematology

6. LPS induces the autocrine secretion of TNF-, which produces apoptosis
LPS induces the autocrine secretion of TNF-, which produces apoptosis.(A) LPS induces the mRNA expression of TNF-α.
LPS induces the autocrine secretion of TNF-, which produces apoptosis.(A) LPS induces the mRNA expression of TNF-α. Total RNA (20 μg per lane) from macrophages was treated with 100 ng/mL of LPS for the indicated times was analyzed by Northern blotting. (B) LPS induces the secretion of TNF-α. The concentration of TNF-α in the culture supernatants was analyzed by ELISA. Each experiment was performed 3 times and represented as the mean value ± SD. (C) TNF-α induces apoptosis in bone marrow macrophages. Macrophages were stimulated for 12 hours with the indicated concentrations of rmTNF-α. Induction of apoptosis was measured by ELISA. (D) Time course of rmTNF-α–induced apoptosis. Macrophages were treated with rmTNF-α (100 ng/mL) for the indicated periods of time. Each experiment was performed in triplicate and represented as the mean ± SD, and 1 of 3 independent experiments is shown in this figure.
Jordi Xaus et al. Blood 2000;95:
©2000 by American Society of Hematology

7. LPS-induced apoptosis in macrophages from TNF-R KO mice
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.
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.
Jordi Xaus et al. Blood 2000;95:
©2000 by American Society of Hematology

8. LPS-induced apoptosis in macrophages from iNOS KO mice
LPS-induced apoptosis in macrophages from iNOS KO mice.(A) LPS induces TNF-α expression in iNOS KO macrophages.
LPS-induced apoptosis in macrophages from iNOS KO mice.(A) LPS induces TNF-α expression in iNOS KO macrophages. The expression of iNOS and TNF-α mRNA was analyzed by Northern blotting. (B) LPS induces TNF-α expression in iNOS KO macrophages. The secretion of TNF-α was analyzed by ELISA in macrophage cultures from each group of mice. Each experiment was performed 3 times and represented as the mean value ± SD. (C) LPS induced similar rates of apoptosis in macrophages from iNOS KO and 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.
Jordi Xaus et al. Blood 2000;95:
©2000 by American Society of Hematology

9. NO-dependent, but not TNF-–dependent, LPS-induced apoptosis was due to p53 and Bax expression.Macrophages from control mice were treated with LPS (100 ng/mL) for the indicated periods of time.
NO-dependent, but not TNF-–dependent, LPS-induced apoptosis was due to p53 and Bax expression.Macrophages from control mice were treated with LPS (100 ng/mL) for the indicated periods of time. Expression of p53 was analyzed by Western blotting (A), whereas expression of Bax mRNA was analyzed by Northern blotting (B). The expression of p53 (C) and Bax (D) was analyzed in macrophages treated with either 50 μmol/L SNAP or 100 ng/mL rmTNF-α for 24 hours. Northern and Western blotting were performed as described in the “Materials and methods” section.
Jordi Xaus et al. Blood 2000;95:
©2000 by American Society of Hematology