Fig 1.
XRCC1 expression during macrophage maturation and poly(ADP-ribose) formation after hydrogen peroxide treatment in monocytes and monocyte-derived macrophages.
(A) Representative images of immunofluorescence staining of XRCC1 in monocytes differentiating into macrophages through GM-CSF treatment over a period of 6 days. (B) Quantification of the mean fluorescence signal of XRCC1. Each dot represents the fluorescence intensity of a single cell. (C) Representative images of the immunofluorescence staining of PAR in monocytes and monocytes that were differentiated into macrophages (at day 3). The control displayed no PAR signal whereas cells treated with 1 mM H2O2 for 5 min showed increased levels of PAR in macrophages on day 3 of differentiation. (D) Quantification of the mean fluorescence signal of PAR. Each dot represents the fluorescence intensity of a single cell. Data are from two independent experiments with at least 50 cells counted for each sample ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, ****p < 0.0001.
Fig 2.
(A) Cells were stained with 10 μM CM-H2DCFDA immediately before treatment with PMA or t-BOOH for 30 min. PMA triggered intracellular ROS production similar to the positive control t-BOOH. (B) Extracellular ROS produced by monocytes and macrophages following treatment with PMA was measured via chemiluminescence with luminol plus HRP and then quantified. (C) The extracellular ROS production of monocytes and macrophages measured over time showed different kinetics. PMA was added to the cells at zero time and chemiluminescence was measured thereafter as described. Data are the mean of at least three independent experiments ± SEM, 1-way ANOVA, Dunnett's Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3.
8-oxo-guanine (8OHdG) formation, DNA break induction and apoptosis in monocytes and macrophages following ROS burst resulting from PMA treatment.
(A) The initial oxidative DNA damage was measured in monocytes and macrophages using an FPG-modified alkaline Comet assay. Cells were treated with PMA for 15 min and then incubated for additional 45 min in PMA free medium. Monocytes and macrophages showed similar levels of initial DNA damage. (B) In parallel, 8OHdG was detected via immunostaining. Both monocytes and macrophages displayed clear 8OHdG staining after PMA treatment compared to the solvent control. (C) DNA strand breaks were measured at different times after the ROS burst was induced by 15 min PMA treatment. Monocytes displayed increasing levels of strand breaks over time compared to macrophages. (D) Monocytes and macrophages were treated with PMA for 15 min and cell death was measured 48 h later. Monocytes displayed increased apoptosis compared to macrophages, which were resistant. Control, untreated cells; solvent, DMSO control (see material and methods). Data are the mean of at least three independent experiments ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, **p < 0.01, ***p < 0.001.
Fig 4.
Monocytes in a co-culture setting with activated macrophages display oxidative DNA damage.
(A) Monocytes were co-cultured with solvent-treated (Mph-Solvent) or PMA-activated (Mph-PMA) macrophages for 1 h before 8OHdG was detected. Monocytes exposed to activated macrophages displayed increased 8OHdG signals. (B) 1 h after co-culture with activated macrophages monocytes were analysed for oxidative DNA damage using the FPG-modified alkaline Comet assay. Monocytes exposed to PMA-activated macrophages (Mph-PMA) displayed increased DNA damage. Data are the mean of four independent experiments ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, **p < 0.01.
Fig 5.
The DNA damage response was activated in monocytes co-cultured with activated macrophages for 24 h.
(A) Monocytes displayed pATM immunostaining when they were co-cultured with activated macrophages (Mph-PMA), but not with non-activated macrophages (Mph-Solvent). (B) Monocytes showed increased 53BP1 foci formation after co-culture with activated macrophages. (C) There was stabilisation of p53 protein as well as phosphorylation of p53 at position Ser46 and phosphorylation of CHK2 in monocytes exposed to activated macrophages (Mph-PMA).
Fig 6.
Apoptosis of monocytes and macrophages co-cultured with non-activated and activated macrophages.
(A) Monocytes were co-cultured with un-treated (Control), solvent-treated (Mph-Solvent) or PMA-activated (Mph-PMA) macrophages and apoptosis was measured 48 h later. Monocytes and macrophages were also concomitantly exposed to PMA (PMA) and apoptosis was measured after 48 h. Exposure of both cell types to PMA exacerbated the killing effect in monocytes. (B) Macrophages themselves showed in the same experimental setting no toxicity after short-term treatment with PMA (Mph-PMA) and only a moderate increase in cell death after direct exposure to PMA (PMA). (C) Monocytes were co-cultured with solvent-treated or PMA-activated macrophages in the absence or presence of the ROS scavenger DMTU (10 mM). In the presence of DMTU, they showed significantly less cell death than monocytes co-cultured with macrophages in the absence of ROS scavenger. Data are the mean of at least four independent experiments ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 7.
LPS/bzATP-activated macrophages and PMA-activated granulocytes produce ROS that kills monocytes in co-culture.
(A) ROS production in LPS/bzATP-activated macrophages. LPS/bzATP was added to the cells at zero time. (B) Apoptosis of monocytes co-cultured for 24 h with LPS/bzATP-activated macrophages. (C) Extracellular ROS produced by granulocytes as a function of time following addition of PMA to the medium. The kinetics were similar to ROS produced by activated monocytes. T cells are shown for comparison; they do not produce ROS. (D) Monocytes were co-cultured with solvent-treated (Granulo-Solvent) or PMA-activated granulocytes (Granulo-PMA) and apoptosis was measured in monocytes after 48 h. Data are the mean of four independent experiments ± SEM, 1-way ANOVA, Tukey’s Multiple Comparison Test, ***p < 0.001.
Fig 8.
Maturation of monocytes by GM-CSF after co-cultivation with PMA-activated macrophages.
(A) Representative images of monocytes cultivated after co-culture with macrophages in the presence of GM-CSF. Monocytes were co-cultured with untreated (Control), solvent-treated (Mph-Solvent) or PMA-stimulated macrophages (Mph-PMA). Thereafter monocytes were separated from macrophages, re-seeded and differentiated into macrophages by adding GM-CSF. On day 6 the generated macrophages were analysed. (B) Analysis of the cell population on day 6 showed a high level of apoptosis (Annexin V+) in cells previously co-cultured with PMA-stimulated macrophages (Mph-PMA) or directly treated with PMA for 24 h (PMA). Data are the mean of at least three independent experiments ± SEM, 1-way ANOVA, Bonferroni's Multiple Comparison Test, **p < 0.01, ***p < 0.001.
Fig 9.
Model of regulation of the immune response by killing of monocytes in trans.
Since monocytes are not only precursors of macrophages but also dendritic cells (DC), their selective killing may also have an impact on the amount of DCs and DC-mediated responses during infection and inflammation.