Decrease in cellular proliferation observed with the removal of serum (and 2-ME) was not due to decreased cell viability as evidenced by the fact that relative to their counterparts cultured in the presence of serum, THP-1 cells cultured in the absence of serum exhibited higher metabolic activity and faster differentiation. PMA-differentiated THP-1 cells cultured in the absence of serum also released approximately 30 less b-hexosaminidase, which correlated with significantly greater retention of b-hexosaminidase in the intracellular compartment.Thus, one of the most striking findings of our study was that 1531364 the removal of serum from the culture medium for 48?6 hours enhanced THP-1 cell viability and function. Hypoxic conditions have been shown to profoundly affect a broad range of myeloid cell properties in vitro, including expression of chemokine receptors and other cell-surface proteins, cytokine secretion, adhesion, migration, phagocytosis and cell survival [33,34]. Thus, it is perhaps not surprising that lowering the oxygen tension from hyperoxic levels to normoxic levels altered macrophage functions in differentiated THP-1 cells. It has been reported that macrophages constitutively release small amounts of b-hexosaminidase independent of external stimuli [22,23]. This continuous low-level release is important for maintaining the normal turnover of glycosaminoglycan in the tissue matrix; however, the release of hexosaminidases increases significantly during an inflammatory event and this contributes to the degradation of the surrounding tissue [35]. Differentiated THP-1 cells continuously release b-hexosaminidase under all the culture conditions tested, but the amount of enzyme released is significantly influenced by not only the removal of serum and 2ME, as discussed above, but also by oxygen tension. Decreasing the oxygen tension to 5 O2 decreased b-hexosaminidase release coincident with increased intracellular levels of the enzyme. These data suggest that under normoxic conditions, the cells are better primed for responding to an inflammatory signal. Phagocytosis by macrophages is an essential component of innate immunity. Previous studies have Eliglustat cost demonstrated that oxygen tension influences this activity. Pfau and collegues [36] demonstrated that peritoneal macrophages, which are exposed to normoxic conditions, and bone-derived macrophages cultured under low oxygen tension exhibed increased phagocytic activity relative to alveolar macrophages, which are exposed in vivo to hyperoxic conditions, and bone marrow-derived macrophages cultured under high oxgen tension. In contrast, there are numerous reports that alveolar macrophages have greater functional activity related to antimicrobial defense, including phagocytosis, compared to 24786787 interstitial macrophages which display enhanced capabilities relevant to specific immune responses such as antigen processing as well as in antioxidant defenses [28,37,38]. Additional studies observed that hypoxia, in vivo and in vitro, increase the phagocytic activity of macrophages [39]. TheseOxygen Tension Influences THP-1 Cell Physiologyapparent discrepancies may be attributed in part to the fact that phagocytosis is positively regulated by hypoxia-inducible factor-1a (HIF1a) [39], which is upregulated by hypoxia [40], and hypoxic conditions are typically associated with inflammatory processes [34]. In contrast, under normoxic conditions, Clavulanic acid potassium salt price HIF-1a is ubiquitinated and therefore less active [41]. Our data p.Decrease in cellular proliferation observed with the removal of serum (and 2-ME) was not due to decreased cell viability as evidenced by the fact that relative to their counterparts cultured in the presence of serum, THP-1 cells cultured in the absence of serum exhibited higher metabolic activity and faster differentiation. PMA-differentiated THP-1 cells cultured in the absence of serum also released approximately 30 less b-hexosaminidase, which correlated with significantly greater retention of b-hexosaminidase in the intracellular compartment.Thus, one of the most striking findings of our study was that 1531364 the removal of serum from the culture medium for 48?6 hours enhanced THP-1 cell viability and function. Hypoxic conditions have been shown to profoundly affect a broad range of myeloid cell properties in vitro, including expression of chemokine receptors and other cell-surface proteins, cytokine secretion, adhesion, migration, phagocytosis and cell survival [33,34]. Thus, it is perhaps not surprising that lowering the oxygen tension from hyperoxic levels to normoxic levels altered macrophage functions in differentiated THP-1 cells. It has been reported that macrophages constitutively release small amounts of b-hexosaminidase independent of external stimuli [22,23]. This continuous low-level release is important for maintaining the normal turnover of glycosaminoglycan in the tissue matrix; however, the release of hexosaminidases increases significantly during an inflammatory event and this contributes to the degradation of the surrounding tissue [35]. Differentiated THP-1 cells continuously release b-hexosaminidase under all the culture conditions tested, but the amount of enzyme released is significantly influenced by not only the removal of serum and 2ME, as discussed above, but also by oxygen tension. Decreasing the oxygen tension to 5 O2 decreased b-hexosaminidase release coincident with increased intracellular levels of the enzyme. These data suggest that under normoxic conditions, the cells are better primed for responding to an inflammatory signal. Phagocytosis by macrophages is an essential component of innate immunity. Previous studies have demonstrated that oxygen tension influences this activity. Pfau and collegues [36] demonstrated that peritoneal macrophages, which are exposed to normoxic conditions, and bone-derived macrophages cultured under low oxygen tension exhibed increased phagocytic activity relative to alveolar macrophages, which are exposed in vivo to hyperoxic conditions, and bone marrow-derived macrophages cultured under high oxgen tension. In contrast, there are numerous reports that alveolar macrophages have greater functional activity related to antimicrobial defense, including phagocytosis, compared to 24786787 interstitial macrophages which display enhanced capabilities relevant to specific immune responses such as antigen processing as well as in antioxidant defenses [28,37,38]. Additional studies observed that hypoxia, in vivo and in vitro, increase the phagocytic activity of macrophages [39]. TheseOxygen Tension Influences THP-1 Cell Physiologyapparent discrepancies may be attributed in part to the fact that phagocytosis is positively regulated by hypoxia-inducible factor-1a (HIF1a) [39], which is upregulated by hypoxia [40], and hypoxic conditions are typically associated with inflammatory processes [34]. In contrast, under normoxic conditions, HIF-1a is ubiquitinated and therefore less active [41]. Our data p.
