Ion raised by the differential effects of 18 versus 5 oxygen tension on LPS-induced NF-kB activation is whether this reflects Title Loaded From File differences in cellular ROS levels since NF-kB is a redox-responsive transcriptional factor [29]. To address this question, we determined whether by pretreating cells with inhibitors of ROS-generating sources (i.e., NADPH oxidase and lipoxygenase) would attenuate LPS-induced NF-kB activation and whether this attenuation would vary in magnitude between cultures grown under 18 versus 5 O2. Differentiated THP-1 cells were cultured under different oxygen tensions in the absence or presence of varying concentrations of the diphenylene iodinium (DPI), an NADPH oxidase inhibitor or nordihydroguaiaretic acid (NGA), an inhibitor of lipoxygenase, for 4 h followed by LPS stimulation for 24 h. Both DPI and NGA significantly inhibited LPS-induced NF-kB activation in a concentration-dependent manner in THP-1 cells grown under either oxygen tension, although a significantly greater inhibition was observed in cultures grown under 18 O2 relative to cultures grown under 5 O2 (Fig. 7A and B).Oxygen Tension Influences THP-1 Cell PhysiologyFigure 6. Oxygen tension influences LPS-induced NF-kB activation and release of cytokines in PMA-differentiated THP-1 cells. Undifferentiated THP-1 XBlue cells, which express an NF-kB reporter gene linked to secreted embryonic alkaline phosphatase (SEAP) were synchronized by serum deprivation for 48 h, and then differentiated with PMA (20 ng/ml) for 48 h in the absence of 2-ME and FBS. Differentiated THP-1 XBlue cells were then cultured in the absence (baseline) or presence of LPS (1 mg/ml) for an additional 24 h in either 18 (A, C) or 5 (B, D) O2. SEAP activity was quantified by QuantiBlue at 630 nm (A,B). Conditioned media from these cultures were analyzed using a human Milliplex KitH to simultaneously quantify multiple cytokines and chemokines released from differentiated THP-1 cell during the 24 h incubation. Each symbol in panels C and D represents the mean of duplicates from one of five wells run in a representative experiment. Data are presented as the mean 6 SEM (n = 2 independent experiments). ***Significantly different from baseline under the same oxygen tension at p,0.001, ###significantly different from 18 O2 at p,0.001 (Student’s Title Loaded From File t-test). doi:10.1371/journal.pone.0054926.gOxygen UptakePericellular pO2, which is a primary determinant of oxygendependent cellular responses, is influenced by both atmospheric oxygen and the oxygen consumption of the cells. Thus, we used a Clark-type O2 electrode to determine whether 1662274 mitochondrial oxygen consumption varied in differentiated THP-1 cells grown under 18 versus 5 O2 for 48 h after synchronization. The rate of mitochondrial oxygen consumption in cells grown under 5 O2 was 0.55 nmol O26(min606 cells)21; whereas cells grown under 18 O2 exhibited a slightly higher rate of oxygen consumption of 0.62 nmol O26(min6106 cells)21. Under both oxygen tensions, rates of oxygen consumption were inhibited by more than 90 by addition of oligomycin (data not shown), indicating that most, if not all, oxygen uptake was linked to oxidative phosphorylation(i.e., mitochondrial ATP production). The ratio of the steady-state concentrations of oxygen around the cells grown at 18 vs. 5 was 3.8 as calculated using the cellular rates of oxygen uptake and ?the experimental concentration of oxygen in growth media at 20C which is 262.2 mM. This ratio of 3.8 is.Ion raised by the differential effects of 18 versus 5 oxygen tension on LPS-induced NF-kB activation is whether this reflects differences in cellular ROS levels since NF-kB is a redox-responsive transcriptional factor [29]. To address this question, we determined whether by pretreating cells with inhibitors of ROS-generating sources (i.e., NADPH oxidase and lipoxygenase) would attenuate LPS-induced NF-kB activation and whether this attenuation would vary in magnitude between cultures grown under 18 versus 5 O2. Differentiated THP-1 cells were cultured under different oxygen tensions in the absence or presence of varying concentrations of the diphenylene iodinium (DPI), an NADPH oxidase inhibitor or nordihydroguaiaretic acid (NGA), an inhibitor of lipoxygenase, for 4 h followed by LPS stimulation for 24 h. Both DPI and NGA significantly inhibited LPS-induced NF-kB activation in a concentration-dependent manner in THP-1 cells grown under either oxygen tension, although a significantly greater inhibition was observed in cultures grown under 18 O2 relative to cultures grown under 5 O2 (Fig. 7A and B).Oxygen Tension Influences THP-1 Cell PhysiologyFigure 6. Oxygen tension influences LPS-induced NF-kB activation and release of cytokines in PMA-differentiated THP-1 cells. Undifferentiated THP-1 XBlue cells, which express an NF-kB reporter gene linked to secreted embryonic alkaline phosphatase (SEAP) were synchronized by serum deprivation for 48 h, and then differentiated with PMA (20 ng/ml) for 48 h in the absence of 2-ME and FBS. Differentiated THP-1 XBlue cells were then cultured in the absence (baseline) or presence of LPS (1 mg/ml) for an additional 24 h in either 18 (A, C) or 5 (B, D) O2. SEAP activity was quantified by QuantiBlue at 630 nm (A,B). Conditioned media from these cultures were analyzed using a human Milliplex KitH to simultaneously quantify multiple cytokines and chemokines released from differentiated THP-1 cell during the 24 h incubation. Each symbol in panels C and D represents the mean of duplicates from one of five wells run in a representative experiment. Data are presented as the mean 6 SEM (n = 2 independent experiments). ***Significantly different from baseline under the same oxygen tension at p,0.001, ###significantly different from 18 O2 at p,0.001 (Student’s t-test). doi:10.1371/journal.pone.0054926.gOxygen UptakePericellular pO2, which is a primary determinant of oxygendependent cellular responses, is influenced by both atmospheric oxygen and the oxygen consumption of the cells. Thus, we used a Clark-type O2 electrode to determine whether 1662274 mitochondrial oxygen consumption varied in differentiated THP-1 cells grown under 18 versus 5 O2 for 48 h after synchronization. The rate of mitochondrial oxygen consumption in cells grown under 5 O2 was 0.55 nmol O26(min606 cells)21; whereas cells grown under 18 O2 exhibited a slightly higher rate of oxygen consumption of 0.62 nmol O26(min6106 cells)21. Under both oxygen tensions, rates of oxygen consumption were inhibited by more than 90 by addition of oligomycin (data not shown), indicating that most, if not all, oxygen uptake was linked to oxidative phosphorylation(i.e., mitochondrial ATP production). The ratio of the steady-state concentrations of oxygen around the cells grown at 18 vs. 5 was 3.8 as calculated using the cellular rates of oxygen uptake and ?the experimental concentration of oxygen in growth media at 20C which is 262.2 mM. This ratio of 3.8 is.
