Figure 3
Figure 3. Proinflammatory cytokines induce neutrophil-to-monocyte reprogramming. (A) Primary G-CSF–induced neutrophilic granulocytes (untreated) were stimulated with G-CSF (100 ng/mL, lane 2) or proinflammatory cytokines (GM-CSF 10 ng/mL, TNFα 25 ng/mL) plus IL-1β (10 ng/mL; +IL-1β, lane 3) or IL-4 (25 ng/mL; +IL-4, lane 4). After 72 hours, whole-cell lysates were prepared for western blot analysis. (B) Neutrophils generated as in Figure 1 were stimulated with GM-CSF (10 ng/mL), TNFα (25 ng/mL), and IL-1β (10 ng/mL) as indicated, and whole-cell extracts were analyzed for c-Jun and C/EBPα protein levels. (C) Neutrophils isolated from G-CSF–mobilized blood display band-stage neutrophil morphology and are devoid of detectable CD14+ and CD34+ cells. Freshly isolated neutrophils from G-CSF–mobilized donors were analyzed by FACS for neutrophil markers (CD15 and LF), the hematopoietic stem/progenitor cell marker CD34, and the monocyte marker CD14. Quadrants were set according to isotype control stainings. (D) Neutrophils from G-CSF–mobilized, but not normal, donors show a monocytic phenotype in response to proinflammatory cytokines. After 5 days of stimulation with proinflammatory cytokines (GM-CSF 10 ng/mL, TNFα 25 ng/mL, IL-1β 10 ng/mL) or G-CSF (100 ng/mL), CD15+ cells were analyzed for CD14 vs LF expression by FACS. Bars represent mean ± SD (percentages of phenotypically defined cells; n = 4 normal and n = 4 G-CSF mobilized donors; ***P < 0.001, **P < 0.005). (E) Assessment of cell proliferation of neutrophils in culture. Neutrophils from G-CSF–mobilized donors were labeled with PKH26 (day 0) and further cultivated in the presence of G-CSF (100 ng/mL) or proinflammatory cytokines (GM-CSF 10 ng/mL, TNFα 25 ng/mL, IL-1β 10 ng/mL) for 5 days. Data are representative of 3 independent experiments. Bars represent mean percent cell recovery at day 5 after culture of neutrophils in the presence of the indicated cytokines (n = 4). (F) Day 5–stimulated CD14hi cells were separated by magnetic-activated cell sorting (MACS) and analyzed for c-Jun protein expression. (G) Neutrophils from G-CSF–mobilized blood (PMN), MACS-sorted day 5–stimulated CD14hi cells (CD14hi), and MACS-sorted monocytes from normal blood (Mo) were analyzed for arginase-1 and C/EBPα expression by RT-PCR. Mean values ± SD were calculated from 3 independent experiments and donors (*P < .05, ***P < .0001).

Proinflammatory cytokines induce neutrophil-to-monocyte reprogramming. (A) Primary G-CSF–induced neutrophilic granulocytes (untreated) were stimulated with G-CSF (100 ng/mL, lane 2) or proinflammatory cytokines (GM-CSF 10 ng/mL, TNFα 25 ng/mL) plus IL-1β (10 ng/mL; +IL-1β, lane 3) or IL-4 (25 ng/mL; +IL-4, lane 4). After 72 hours, whole-cell lysates were prepared for western blot analysis. (B) Neutrophils generated as in Figure 1 were stimulated with GM-CSF (10 ng/mL), TNFα (25 ng/mL), and IL-1β (10 ng/mL) as indicated, and whole-cell extracts were analyzed for c-Jun and C/EBPα protein levels. (C) Neutrophils isolated from G-CSF–mobilized blood display band-stage neutrophil morphology and are devoid of detectable CD14+ and CD34+ cells. Freshly isolated neutrophils from G-CSF–mobilized donors were analyzed by FACS for neutrophil markers (CD15 and LF), the hematopoietic stem/progenitor cell marker CD34, and the monocyte marker CD14. Quadrants were set according to isotype control stainings. (D) Neutrophils from G-CSF–mobilized, but not normal, donors show a monocytic phenotype in response to proinflammatory cytokines. After 5 days of stimulation with proinflammatory cytokines (GM-CSF 10 ng/mL, TNFα 25 ng/mL, IL-1β 10 ng/mL) or G-CSF (100 ng/mL), CD15+ cells were analyzed for CD14 vs LF expression by FACS. Bars represent mean ± SD (percentages of phenotypically defined cells; n = 4 normal and n = 4 G-CSF mobilized donors; ***P < 0.001, **P < 0.005). (E) Assessment of cell proliferation of neutrophils in culture. Neutrophils from G-CSF–mobilized donors were labeled with PKH26 (day 0) and further cultivated in the presence of G-CSF (100 ng/mL) or proinflammatory cytokines (GM-CSF 10 ng/mL, TNFα 25 ng/mL, IL-1β 10 ng/mL) for 5 days. Data are representative of 3 independent experiments. Bars represent mean percent cell recovery at day 5 after culture of neutrophils in the presence of the indicated cytokines (n = 4). (F) Day 5–stimulated CD14hi cells were separated by magnetic-activated cell sorting (MACS) and analyzed for c-Jun protein expression. (G) Neutrophils from G-CSF–mobilized blood (PMN), MACS-sorted day 5–stimulated CD14hi cells (CD14hi), and MACS-sorted monocytes from normal blood (Mo) were analyzed for arginase-1 and C/EBPα expression by RT-PCR. Mean values ± SD were calculated from 3 independent experiments and donors (*P < .05, ***P < .0001).

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