Arallel these findings in that we observed that differentiated THP-1 cells exhibited increased phagocytosis when cultured under hyperoxic (18 O2) conditions relative to normoxic (5 O2) conditions. This result is also consistent with evidence that phagocytosis is dependent on the respiratory burst, which requires extracellular oxygen [27]. Collectively, these data implicate O2 or a metabolite in the acute regulation of phagocytosis. We tested this possibility by switching the oxygen tension under which differentiated THP-1 cells were maintained during the last hour of a 25 h incubation period. Regardless of 1531364 the oxygen tension under which THP-1 cells were originally cultured, phagocytic activity was predominantly influenced by the oxygen tension during the specific time of phagocytosis, thus linking control of phagocytosis to environmental oxygen tension. It is well documented that NF-kB activation requires an oxidative burst, which releases it from IkB [42]. Therefore, it was not particularly surprising that while LPS stimulated NF-kB in differentiated THP-1 cells grown under either oxygen condition, this response was attenuated in cells exposed to 5 O2 relative to 18 O2. NF-kB activation is closely linked to cytokine release in (��)-Hexaconazole chemical information macrophages [43], so we predicted that LPS-stimulated release of cytokines would similarly be attenuated by culture in 5 O2. However, as indicated by multiplex cytokine analyses, differentiated THP-1 cells cultured in 5 O2 exhibited a more robust increase in cytokine release upon LPS stimulation relative to baseline than cells cultured in 18 O2. The concentrations of cytokines released upon LPS stimulation were comparable Cucurbitacin I cost between cultures exposed to either oxygen tension, so the increased differential between baseline and LPS-induced levels of cytokine secretion in cells cultured under 5 O2 reflect the fact that the basal levels of cytokine secretion in the absence of LPS were significantly lower in these cultures relative to those exposed to 18 O2. Although the NF-kB activation baseline was not changed at 5 O2, these results from the cytokine release studies are consistent with results from the antioxidant studies showing enhanced inhibition of LPS-induced NF-kB activation at 18 O2 versus 5 O2, suggesting a more pronounced role for ROS signaling in THP-1 cells grown under 18 O2.unprecedented, and extensive and efficient use of this approach has been made in specific tissue culture models, including cultures of placental explant [44], embryos [45] and stem cells [2]. The present study confirms a major role for extracellular oxygen tension in modulating THP-1 cell physiology, which is consistent with literature documenting the regulation of immune function by the cellular redox environment both in 24786787 vivo and in primary human monocytes and macrophages [46]. Our findings also suggest that THP-1 cells grown under tightly regulated oxygen tension in the absence of exogenous reducing agent are likely to provide a more physiologically relevant baseline from which to study the role of the local redox environment in regulating macrophage differentiation and function.Materials and Methods Cell CultureHuman monocytic THP-1 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) [1]. THP1XBlue cells, which were derived from the human monocytic THP1 cell line, were purchased from Invitrogen (San Diego, CA). THP1-XBlue cells are NF-kB-reporter cells in which activation of the transcription f.Arallel these findings in that we observed that differentiated THP-1 cells exhibited increased phagocytosis when cultured under hyperoxic (18 O2) conditions relative to normoxic (5 O2) conditions. This result is also consistent with evidence that phagocytosis is dependent on the respiratory burst, which requires extracellular oxygen [27]. Collectively, these data implicate O2 or a metabolite in the acute regulation of phagocytosis. We tested this possibility by switching the oxygen tension under which differentiated THP-1 cells were maintained during the last hour of a 25 h incubation period. Regardless of 1531364 the oxygen tension under which THP-1 cells were originally cultured, phagocytic activity was predominantly influenced by the oxygen tension during the specific time of phagocytosis, thus linking control of phagocytosis to environmental oxygen tension. It is well documented that NF-kB activation requires an oxidative burst, which releases it from IkB [42]. Therefore, it was not particularly surprising that while LPS stimulated NF-kB in differentiated THP-1 cells grown under either oxygen condition, this response was attenuated in cells exposed to 5 O2 relative to 18 O2. NF-kB activation is closely linked to cytokine release in macrophages [43], so we predicted that LPS-stimulated release of cytokines would similarly be attenuated by culture in 5 O2. However, as indicated by multiplex cytokine analyses, differentiated THP-1 cells cultured in 5 O2 exhibited a more robust increase in cytokine release upon LPS stimulation relative to baseline than cells cultured in 18 O2. The concentrations of cytokines released upon LPS stimulation were comparable between cultures exposed to either oxygen tension, so the increased differential between baseline and LPS-induced levels of cytokine secretion in cells cultured under 5 O2 reflect the fact that the basal levels of cytokine secretion in the absence of LPS were significantly lower in these cultures relative to those exposed to 18 O2. Although the NF-kB activation baseline was not changed at 5 O2, these results from the cytokine release studies are consistent with results from the antioxidant studies showing enhanced inhibition of LPS-induced NF-kB activation at 18 O2 versus 5 O2, suggesting a more pronounced role for ROS signaling in THP-1 cells grown under 18 O2.unprecedented, and extensive and efficient use of this approach has been made in specific tissue culture models, including cultures of placental explant [44], embryos [45] and stem cells [2]. The present study confirms a major role for extracellular oxygen tension in modulating THP-1 cell physiology, which is consistent with literature documenting the regulation of immune function by the cellular redox environment both in 24786787 vivo and in primary human monocytes and macrophages [46]. Our findings also suggest that THP-1 cells grown under tightly regulated oxygen tension in the absence of exogenous reducing agent are likely to provide a more physiologically relevant baseline from which to study the role of the local redox environment in regulating macrophage differentiation and function.Materials and Methods Cell CultureHuman monocytic THP-1 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA) [1]. THP1XBlue cells, which were derived from the human monocytic THP1 cell line, were purchased from Invitrogen (San Diego, CA). THP1-XBlue cells are NF-kB-reporter cells in which activation of the transcription f.
