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Apoptosis is essential for neutrophil functional shutdown and determines tissue damage in experimental pneumococcal meningitis.

Koedel U, Frankenberg T, Kirschnek S, Obermaier B, Häcker H, Paul R, Häcker G - PLoS Pathog. (2009)

Bottom Line: We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis.The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1beta and G-CSF as well as reduced levels of anti-inflammatory TGF-beta.In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Clinic of the University of Munich, Munich, Germany.

ABSTRACT
During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1beta and G-CSF as well as reduced levels of anti-inflammatory TGF-beta. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils.

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Overexpression of Bcl-2 protects neutrophils from apoptosis and removal by macrophages.(A) wt or Bcl-2-overexpressing neutrophils differentiated for 5 days were in addition cultured for 24 or 48 hours in the presence or absence of SCF. Cells were then stained with AnnexinV-FITC and propidium iodide (PI) and analysed by flow cytometry. Bars/error bars indicate mean/SEM of five independent experiments. (B) Neutrophils differentiated for 5 days were cultured for 8 or 48 hours in the presence or absence of SCF, followed by staining for active caspase-3. Error bars show the SEM of three independent experiments. (C) Neutrophils differentiated for 5 days were cultured in the absence of SCF for the indicated periods of time and stained with AnnexinV-PI or CFSE. Green-fluorescent (CFSE-stained) neutrophils were then added to cultures of RAW macrophages stained with the red dye PKH26 (ratio neutrophil∶macrophages, 5∶1). After 4 h of co-incubation, cultures were analysed by flow cytometric analysis. The proportion of phagocytic cells within the macrophage population is shown for wt or Bcl-2-expressing neutrophils at various time points. No double-positive cells were observed when reactions were carried out at 4°C (data not shown). Data are mean/SEM from 3 independent experiments.
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ppat-1000461-g003: Overexpression of Bcl-2 protects neutrophils from apoptosis and removal by macrophages.(A) wt or Bcl-2-overexpressing neutrophils differentiated for 5 days were in addition cultured for 24 or 48 hours in the presence or absence of SCF. Cells were then stained with AnnexinV-FITC and propidium iodide (PI) and analysed by flow cytometry. Bars/error bars indicate mean/SEM of five independent experiments. (B) Neutrophils differentiated for 5 days were cultured for 8 or 48 hours in the presence or absence of SCF, followed by staining for active caspase-3. Error bars show the SEM of three independent experiments. (C) Neutrophils differentiated for 5 days were cultured in the absence of SCF for the indicated periods of time and stained with AnnexinV-PI or CFSE. Green-fluorescent (CFSE-stained) neutrophils were then added to cultures of RAW macrophages stained with the red dye PKH26 (ratio neutrophil∶macrophages, 5∶1). After 4 h of co-incubation, cultures were analysed by flow cytometric analysis. The proportion of phagocytic cells within the macrophage population is shown for wt or Bcl-2-expressing neutrophils at various time points. No double-positive cells were observed when reactions were carried out at 4°C (data not shown). Data are mean/SEM from 3 independent experiments.

Mentions: We tested the anti-apoptotic effect of Bcl-2 against spontaneous neutrophil apoptosis in two conditions. Neutrophils were differentiated in vitro for five days in the presence of SCF. At day 5 to 6, wt neutrophils began to undergo spontaneous apoptosis (detected by staining for annexin V and propidium iodide and by nuclear morphology) despite the continued presence of SCF, reaching levels of about 60% apoptotic cells 48 h later (on day 7) (Fig. 3A; Fig. S4A gives an example of the primary data). When SCF was washed away on day 5, the apoptotic population was about 50% on day 6 and 80% on day 7. Under the same conditions, there was hardly any apoptosis detectable on day 6 in Bcl-2 neutrophils, while about 15–25% of cells were apoptotic on day 7. Surprisingly, there was no difference between Bcl-2 neutrophil cultures with and without SCF (Fig. 3A). This suggests that Bcl-2 was able completely to block apoptosis due to SCF-withdrawal but that there was a component of spontaneous apoptosis that was not inhibited by Bcl-2. Apoptosis was accompanied by rapid activation of caspase-3 in wt cells, and Bcl-2 blocked this activation (Fig. 3B and Fig. S4B).


Apoptosis is essential for neutrophil functional shutdown and determines tissue damage in experimental pneumococcal meningitis.

Koedel U, Frankenberg T, Kirschnek S, Obermaier B, Häcker H, Paul R, Häcker G - PLoS Pathog. (2009)

Overexpression of Bcl-2 protects neutrophils from apoptosis and removal by macrophages.(A) wt or Bcl-2-overexpressing neutrophils differentiated for 5 days were in addition cultured for 24 or 48 hours in the presence or absence of SCF. Cells were then stained with AnnexinV-FITC and propidium iodide (PI) and analysed by flow cytometry. Bars/error bars indicate mean/SEM of five independent experiments. (B) Neutrophils differentiated for 5 days were cultured for 8 or 48 hours in the presence or absence of SCF, followed by staining for active caspase-3. Error bars show the SEM of three independent experiments. (C) Neutrophils differentiated for 5 days were cultured in the absence of SCF for the indicated periods of time and stained with AnnexinV-PI or CFSE. Green-fluorescent (CFSE-stained) neutrophils were then added to cultures of RAW macrophages stained with the red dye PKH26 (ratio neutrophil∶macrophages, 5∶1). After 4 h of co-incubation, cultures were analysed by flow cytometric analysis. The proportion of phagocytic cells within the macrophage population is shown for wt or Bcl-2-expressing neutrophils at various time points. No double-positive cells were observed when reactions were carried out at 4°C (data not shown). Data are mean/SEM from 3 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682662&req=5

ppat-1000461-g003: Overexpression of Bcl-2 protects neutrophils from apoptosis and removal by macrophages.(A) wt or Bcl-2-overexpressing neutrophils differentiated for 5 days were in addition cultured for 24 or 48 hours in the presence or absence of SCF. Cells were then stained with AnnexinV-FITC and propidium iodide (PI) and analysed by flow cytometry. Bars/error bars indicate mean/SEM of five independent experiments. (B) Neutrophils differentiated for 5 days were cultured for 8 or 48 hours in the presence or absence of SCF, followed by staining for active caspase-3. Error bars show the SEM of three independent experiments. (C) Neutrophils differentiated for 5 days were cultured in the absence of SCF for the indicated periods of time and stained with AnnexinV-PI or CFSE. Green-fluorescent (CFSE-stained) neutrophils were then added to cultures of RAW macrophages stained with the red dye PKH26 (ratio neutrophil∶macrophages, 5∶1). After 4 h of co-incubation, cultures were analysed by flow cytometric analysis. The proportion of phagocytic cells within the macrophage population is shown for wt or Bcl-2-expressing neutrophils at various time points. No double-positive cells were observed when reactions were carried out at 4°C (data not shown). Data are mean/SEM from 3 independent experiments.
Mentions: We tested the anti-apoptotic effect of Bcl-2 against spontaneous neutrophil apoptosis in two conditions. Neutrophils were differentiated in vitro for five days in the presence of SCF. At day 5 to 6, wt neutrophils began to undergo spontaneous apoptosis (detected by staining for annexin V and propidium iodide and by nuclear morphology) despite the continued presence of SCF, reaching levels of about 60% apoptotic cells 48 h later (on day 7) (Fig. 3A; Fig. S4A gives an example of the primary data). When SCF was washed away on day 5, the apoptotic population was about 50% on day 6 and 80% on day 7. Under the same conditions, there was hardly any apoptosis detectable on day 6 in Bcl-2 neutrophils, while about 15–25% of cells were apoptotic on day 7. Surprisingly, there was no difference between Bcl-2 neutrophil cultures with and without SCF (Fig. 3A). This suggests that Bcl-2 was able completely to block apoptosis due to SCF-withdrawal but that there was a component of spontaneous apoptosis that was not inhibited by Bcl-2. Apoptosis was accompanied by rapid activation of caspase-3 in wt cells, and Bcl-2 blocked this activation (Fig. 3B and Fig. S4B).

Bottom Line: We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis.The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1beta and G-CSF as well as reduced levels of anti-inflammatory TGF-beta.In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Clinic of the University of Munich, Munich, Germany.

ABSTRACT
During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1beta and G-CSF as well as reduced levels of anti-inflammatory TGF-beta. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils.

Show MeSH
Related in: MedlinePlus