Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

Abstract

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O2 and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O2) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O2 versus 5% O2 indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O2 significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O2 decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.

Quantifying the magnitude of the oxygen artefact inherent in culturing airway cells under atmospheric oxygen versus physiological levels

“…Additionally, Grodzki et al . . demonstrated that THP‐1 macrophages cultured at physiological oxygen tension (5% O 2 ) showed significant decrease in phagocytosis of E. coli particles compared to cells cultured at atmospheric oxygen.…”

Moderate Hypoxia Down-Regulates Interleukin-6 Secretion and TLR4 Expression in Human Sw.71 Placental Cells

“…Similarly, Ishida et al [27] showed that TLR4 mRNA and protein expression was decreased cultured for 48-72 h under hypoxic conditions. In addition, Grodzki et al [28] suggested that THP-1 macrophages are better primed for responding to an inflammatory signal under normoxic conditions because NF-κB activation and secretion of cytokines were attenuated in cells exposed to 5% O 2 relative to 18% O 2 . These findings suggest that moderate hypoxia may have the potential to regulate the inflammatory response via controlling TLR4 expression.…”

Proteomic Signature Reveals Modulation of Human Macrophage Polarization and Functions Under Differing Environmental Oxygen Conditions

“…Furthermore, bone marrow-derived macrophages exposed to low oxygen levels were shown to present an increased rate of bacterial phagocytosis (16). Oxygen exposure also influenced THP1-differentiated macrophages, although by decreasing phagocytosis (17), it therefore appears that oxygen tension is a fundamental modulator of macrophage polarization that should be clarified at the molecular level in human macrophages.…”