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% O₂versus 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.

Figure 1. Influence of O₂ tension, 2-ME and serum on proliferation of undifferentiated THP-1 cells.

Monocytic THP-1 cells were synchronized by serum deprivation for 48 h and then cultured in hyperoxic (18% O₂) or normoxic (5% O₂) with or without 2-ME and/or FBS. Cell density was determined using a hemocytometer at 24 h (A) and 48 h (B) after synchronization. Data are presented as the mean ± SEM (n = 5 independent experiments). *Significantly different from cultures with 2-ME and FBS under the same oxygen tension at p<0.05 (one-way ANOVA with post hoc Tukey’s test).

Figure 2. Influence of O₂ tension, 2-ME and serum on the metabolic activity of THP-1 cells.

THP-1 cells were synchronized by serum deprivation for 48 h. (A) Undifferentiated THP-1 cells were plated in 96-well plates precoated with poly-D-lysine. (B) THP-1 cells were triggered to undergo macrophage differentiation by incubating with PMA at 20 ng/ml for 24 h. MTT was added to both undifferentiated and differentiated THP-1 cells for 3 h under varying O₂, 2-ME and serum conditions. MTT reduction, measured as the absorbance at 562 nm, was normalized to protein concentration. Data are presented as the mean ± SEM (n = 4 independent experiments. *Significantly different from +2-ME+FBS (standard culture conditions) under the same oxygen tension (one-way ANOVA and post hoc Tukey’s test); ^▴Significantly different from –2-ME+FBS under the same oxygen tension (one-way ANOVA and post hoc Tukey’s test); ^#Significantly different from the same culture condition in the 18% O₂ group (e.g., 18% O₂ versus 5% O₂) by Student’s t-test. *, ^#, ^▴ p<0.05; **,^##, ^▴▴ p<0.01; ***, ^###, ^▴▴▴ p<0.001.

Figure 3. Influence of O₂ tension, 2-ME and serum on the rate of THP-1 differentiation.

Differentiation of THP-1 cells from monocytic to macrophage cells is associated with transition from a non-adherent to an adherent cell type. To determine whether culture conditions affect PMA-stimulated differentiation of THP-1 cells to macrophages, cell adhesion was assessed at 3 and 24 h after addition of PMA (20 ng/ml) to the culture medium. Data are presented as the mean ± SEM of the protein concentration of adherent cells at 3 h as a percentage of the protein concentration of adherent cells at 24 h (n = 4 independent experiments). *Significantly different from +2-ME+FBS (standard culture conditions) under the same oxygen tension (one-way ANOVA and post hoc Tukey’s test); ^▴Significantly different from –2-ME+FBS under the same oxygen tension (one-way ANOVA and post hoc Tukey’s test); ^#Significantly different from the same culture condition in the 18% O₂ group (e.g., 18% O₂ versus 5% O₂) by Student’s t-test. **, ^##, ^▴▴ p<0.01; ***, ^###, ^▴▴▴ p<0.001.

Figure 4. Influence of O₂ tension, 2-ME and serum on release of β-hexosaminidase.

Differentiated THP-1 cells constitutively release β-hexosaminidase that is measurable in the conditioned medium (supernatant) after 24 h (A) or 48 h (B) of culture. β-Hexosaminidase is also detected in cell lysates (C). β-Hexosaminidase activity per well was normalized to the concentration of protein in the same well as determined using the BCA protein assay. Data are presented as mean ± SEM (n = 3 independent experiments). *Significantly different from +2-ME+FBS (standard culture conditions) under the same oxygen tension (one-way ANOVA and post hoc Tukey’s test); ^▴Significantly different from –2-ME+FBS under the same oxygen tension (one-way ANOVA and post hoc Tukey’s test); ^#Significantly different from the same culture condition in the 18% O₂ group (e.g., 18% O₂ versus 5% O₂) by Student’s t-test. *, ^#, ^▴ p<0.05; **, ^##, ^▴▴ p<0.01; ***, ^###, ^▴▴▴ p<0.001.

Figure 5. Oxygen tension significantly influences phagocytosis in PMA-differentiated THP-1 cells.

Undifferentiated THP-1 cells were synchronized by serum deprivation for 48 h, plated at a density of 10⁵cells/well in a 96-well plate and differentiated with PMA (20 ng/ml) for 48 h in the absence of 2-ME and FBS. Differentiated THP-1 cells were washed and then incubated for 3 h with E.coli BioParticles®, which emit fluorescence upon acidification in lysosomes following phagocytosis. Phagocytosis, which was quantified by determining the fluorescence intensity at 600 nm, was blocked by pretreating cultures with cytochalasin D (2 µM) for 1 h prior to addition of E. coli BioParticles®. The mean fluorescence intensity was normalized to protein concentration as determined using the BCA protein assay. Data are presented as the mean ± SEM (n = 3 independent experiments). *Significantly different from control (– cytochalasin) treatment under the same oxygen tension; ^#significantly different from the same culture condition in the 18% O₂ treatment group (e.g., 18% O₂ versus 5% O₂) by Student’s t-test. ***, ^### p<0.001.

Figure 6. Oxygen tension influences LPS-induced NF-κB activation and release of cytokines in PMA-differentiated THP-1 cells.

Undifferentiated THP-1 XBlue cells, which express an NF-κB 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 µg/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 Kit® 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 ± 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).

Figure 7. Oxygen tension influences redox in LPS-induced NF-κB activation in PMA-differentiated THP-1 cells.

Undifferentiated THP-1 XBlue cells, which express an NF-κB 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. Differentiated THP-1 XBlue cells were then cultured in varying concentrations of DPI (A) or NGA (B) for 4 h followed by LPS (1 µg/ml) stimulation for an additional 24 h in either 18% or 5% O₂. SEAP activity was quantified by QuantiBlue at 630 nm. Data are presented as the mean ± SEM (n = 2 independent experiments). *Significantly different from baseline (SEAP activity in the absence of inhibitor) at the same oxygen tension; **p<0.01; ***p<0.001 (one-way ANOVA with post hoc Tukey’s test). ^#Significantly different from 5% O₂ at same antioxidant concentration; ^# p<0.05; ^## p<0.01 (Student’s t-test).