Induction of autophagy is essential for monocyte-macrophage differentiation

Abstract

Monocytes are programmed to undergo apoptosis in the absence of stimulation. Stimuli that promote monocyte-macrophage differentiation not only cause cellular changes, but also prevent the default apoptosis of monocytes. In the present study, we demonstrate that autophagy is induced when monocytes are triggered to differentiate and that the induction of autophagy is pivotal for the survival and differentiation of monocytes. We also show that inhibition of autophagy results in apoptosis of cells that are engaged in differentiation. We found that the differentiation signal releases Beclin1 from Bcl-2 by activating JNK and blocks Atg5 cleavage, both of which are critical for the induction of autophagy. Preventing autophagy induction hampers differentiation and cytokine production; therefore, autophagy is an important transition from monocyte apoptosis to differentiation.

Figures

Figure 1

Autophagy is activated during human monocyte differentiation. (A-B) Immunoblot analysis of lysates from human primary elutriated monocytes incubated with or without GM-CSF (10nM) for the indicated times. (B) Where indicated, cells were pretreated with 3-MA at 10mM or CQ at 50μM. Representative data from at least 3 independent experiments for panels A and B are shown. The ratio of LC3II/LC3I was quantified from 3 experiments and is presented in bar graphs (A-B). (C) Human monocytes treated or not with GM-CSF for 14 hours, stained with anti-LC3 Ab and then Alexa Fluor 568 goat anti–mouse IgG (red), and processed for confocal microscopy. Scale bars indicate 5 μm. (D) Human monocytes were incubated with or without GM-CSF for 14 hours and processed for electron microscopy. Black arrows indicate autophagosomes with double membranes and partially degraded material. Scale bars indicate 500 nm. (E) Human monocytes were incubated with or without GM-CSF (10nM) in the absence or presence of 3-MA (10mM) for 14 hours, fixed, and stained with 4′,6-diamidino-2-phenylindole to visualize the nuclei (blue), and immunolabeled with an anti-LC3 Ab followed by 568 goat anti–mouse IgG (red). Scale bars indicate 20 μm. Representative images from 4 independent experiments are shown in panels B through D and quantitation of the percentage of cells with LC3+ punctate from the 4 experiments is shown in panel E (*P < .002 for comparisons between GM-CSF alone and untreated or cotreatment).

Figure 2

The induction of autophagy is observed during monocyte differentiation in vivo. (A) Representative immunoblot analysis of lysates from mouse BM monocytes incubated with or without murine M-CSF at a concentration of 20nM for 14 hours. Where indicated, cells were treated in the presence or absence of 3-MA (10mM) or CQ (50μM). (B) Representative flow analysis of cells from thioglycolate-induced peritonitis costained with F4/80-APC and Ly6C-FITC for the indicated times. (C) Ly6ChighF4/80low BM monocytes and the F4/80+ population from panel B were sorted and their lysates compared by Western blot analysis. Representative immunoblot analyses are shown.

Figure 3

p62 is colocalized with GM-CSF–induced LC3+ puncta and degraded by autophagy in activated monocytes. (A) Human primary monocytes were incubated with or without GM-CSF for 14 hours, and then treated with DQ-BSA for another 2 hours. Cells were fixed and stained with anti-LC3 Ab, followed by an FITC goat anti–mouse IgG (green). Representative confocal images of colocalization of LC3 and red fluorescence of DQ-BSA are shown. (B) Human primary monocytes were pretreated with cycloheximide (CHX) at 1μM or CQ at 50mM, and then incubated with GM-CSF (10nM) for the indicated times and analyzed by immunoblotting against p62. One representative experiment from at least 3 independent experiments is shown. Asterisk indicates one special band of truncated p62 Ab. (C) Human primary monocytes were incubated with or without GM-CSF for 14 hours, and then fixed and costained with anti-p62 Ab followed by a FITC goat anti–mouse IgG (green) and anti-LC3 Ab and then rhodamine goat anti–rabbit IgG (red). Representative confocal images of colocalization of p62 and LC3 are shown. (D) Human primary monocytes were incubated with or without GM-CSF for 14 hours and DQ-BSA for another 2 hours, then fixed and stained with anti-p62 Ab, followed by an FITC goat anti–mouse IgG (green). Representative confocal images of colocalization of p62 and red fluorescence of DQ-BSA are shown. Scale bars indicate 5 μm. Arrows indicate colocalization of green and red fluorescence (A,C,D).

Figure 4

Inhibition of autophagy suppresses GM-CSF–induced human primary monocyte survival. (A) Cell death of human monocytes with different treatments (GM-CSF, 10nM; 3-MA, 10mM; or CQ, 50 mM) for 48 hours was assessed using annexin V/PI staining and flow cytometry. Percentage of annexin V/PI double-negative cells (percentage survival) are shown. Dead cells indicate all cells positive for annexin V, including annexin V+/PI− early apoptosis and annexin V+/PI+ later apoptosis (A,C). Error bars indicate ± SEM. Data are representative of at least 3 independent experiments. (B) Immunoblot analysis with Abs to LC3, Caspase-3, and actin for GM-CSF–treated primary human monocytes. Representative data are shown for GM-CSF alone and pretreatment with 3-MA (10mM) or CQ (50μM). (C) Cell death assessed by flow cytometry of annexin V/PI–stained cells with Beclin1 siRNA or control siRNA in treated or not with GM-CSF. The percentage of annexin V/PI double-negative cells (percentage survival) is shown. Error bare indicate ± SEM. Data are representative of at least 3 independent experiments. *P < .03. (D) Immunoblot analysis of lysates of monocytes transfected with human Beclin1 siRNA or control siRNA probed with anti–Caspase-3, anti-Beclin1, and anti-actin Abs. Data are representative of at least 3 experiments.

Figure 5

Autophagy is required for human primary monocyte differentiation. (A) Morphological changes associated with GM-CSF–induced monocyte/macrophage differentiation from days 1, 3, and 6. Representative phase-contrast images are shown for GM-CSF treatment alone (left panel) and for pretreatment with 3-MA (middle panel) or CQ (right panel). Scale bar indicates 100 μm. (B) Representative FACS plots showing CD14 and CD11b expression in GM-CSF–treated monocytes/macrophages treated or not with 3-MA or CQ. Live Cells were gated and stained with FITC–anti-CD14 Ab and PE–anti-CD11b Ab. (C) Representative FACS plots showing LPS/IFN-γ–stimulated CD86 expression in GM-CSF macrophages pretreated or not with 3-MA or CQ. Gated live cells stained with PE–anti-CD86 Ab are shown. (D) Quantitative real-time PCR analysis of human TNF-α and IL-12p40 mRNA in macrophages pretreated with 3-MA or CQ, presented relative to GAPDH mRNA. P < .005 for comparisons between GM-CSF alone and cotreatment with 3MA or CQ for both graphs. Data are representative of 5 (A) or 3 (B-D) experiments. Histogram shows mean ± SD in panel D.

Figure 6

GM-CSF induces JNK activation to mediate the disassociation of Beclin1 and Bcl-2, the induction of autophagy, and monocyte survival. (A) Immunoblot analysis with anti-LC3 and actin for monocytes pretreated with JNK inhibitor SP (10μM), MEK inhibitor U0126 (10μM), or p38 inhibitor SB (5μM), and then treated with GM-CSF for 14 hours. (B) Immunoblot analysis with Abs to phosphorylated JNK or JNK of monocytes treated with GM-CSF for the indicated times. (C) Human monocytes were treated with or without GM-CSF for the indicated times and lysates were subjected to immunoblot analysis with Abs to phosphorylated Bcl-2 and actin. (D) Western blots of endogenous Beclin1 co-immunoprecipitated by anti–Bcl-2 Ab. (E) Human monocytes pretreated with the JNK inhibitor SP (10μM) in the presence or absence of GM-CSF for the indicated times and lysates were subjected to immunoblot analysis with Abs to phosphorylated Bcl-2, LC3, and Actin. (F) Cell death assessed by annexin V/PI staining showing apoptosis in monocytes untreated or pretreated with JNK inhibitor SP in the presence or absence of GM-CSF for 48 hours. Percentage of annexin V/PI double-negative cells (percentage survival) are shown. Dead cells indicate all cells positive for annexin V, including annexin V+/PI− early apoptosis and annexin V+/PI+ later apoptosis. Error bars indicate± SEM. Data are representative of 3 independent experiments. (G) Lysates were subjected to immunoblotting with Caspase-3 and actin Abs. Western blot data are representative of at least 3 experiments.

Figure 7

GM-CSF blocks the cleavage of apoptosis-related truncated Atg5 by inhibiting calpain activity. (A) Immunoblot analysis with Abs to Atg5 or truncated Atg5 in lysates of human primary monocytes treated or not with GM-CSF. (B) Immunoblot analysis of subcellular fractionations (cytoplasm and mitochondria) of human monocytes that were or were not treated with GM-CSF for 14 hours, probed with anti-truncated Atg5, anti-VADC, and anti–β-Tubulin. (C) Immunoblot analysis with Abs to truncated Atg5 in human primary monocytes treated or not treated with GM-CSF. Where indicated, cells were pretreated with 3-MA (10μM) or CQ (50μM) for the indicated times. (D) Calpain activity assay of lysates from monocytes treated or not with GM-CSF. Error bars indicate ± SEM. Data are representative of 5 independent experiments, *P < .02; **P < .002. (E) Immunoblot analysis with Abs to Atg5 or truncated Atg5 in lysates from human primary monocytes treated or untreated with the calpain inhibitor ZVP (10μM). (F) Immunoblot analysis with Abs to Calpain1, calpastatin, and Actin in lysates of GM-CSF–incubated monocytes. (G) Immunoblot analysis with Abs to LC3, truncated Atg5, Caspase-3, and actin in human primary monocytes lysates treated with GM-CSF and ZVP (10μM). (H) Histogram of cell death assessed by flow cytometry of annexin V/PI–stained cells treated or not with GM-CSF and ZVP for 24 hours. The percentages of annexin V/PI double-negative cells (percentage survival) are shown. Dead cells indicate all cells positive for annexin V, including annexin V+/PI− early apoptosis and annexin V+/PI+ later apoptosis. Error bars indicate ± SEM. Data are representative of at least 3 independent experiments. #P < .02. Western blot data are representative of at least 3 (A-C,E) or 5 (F-G) experiments. Asterisk indicates a nonspecial band of truncated Atg5 Ab (A,C,E,G).