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Regulation of apoptosis and priming of neutrophil oxidative burst by diisopropyl fluorophosphate.

Tsang JL, Parodo JC, Marshall JC - J Inflamm (Lond) (2010)

Bottom Line: Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules.However, DFP enhanced the exteriorization of PS in a dose-dependent manner.We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdepartmental Division of Critical Care, University of Toronto, Toronto, Canada. marshallj@smh.ca.

ABSTRACT

Background: Diisopropyl fluorophosphate (DFP) is a serine protease inhibitor that is widely used as an inhibitor of endogenous proteases in in vitro neutrophil studies. Its effects on neutrophil function are unclear. We sought to determine the biological effects of DFP on human neutrophil apoptosis and oxidative burst.

Methods: We isolated neutrophils from healthy volunteers, incubated them with DFP (2.5 mM), and evaluated neutrophil elastase (NE) activity, neutrophil degranulation, apoptosis as reflected in hypodiploid DNA formation and exteriorization of phosphatidylserine (PS), processing and activity of caspases-3 and -8, oxidative burst activity and hydrogen peroxide release.

Results: Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules. DFP inhibited constitutive neutrophil apoptosis as reflected in DNA fragmentation, and the processing and activity of caspases-3 and -8. DFP also inhibited priming of neutrophils for oxidative burst activity and hydrogen peroxide release. However, DFP enhanced the exteriorization of PS in a dose-dependent manner.

Conclusion: We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity. We further conclude that endogenous proteases play a role in the biology of constitutive neutrophil apoptosis.

No MeSH data available.


Related in: MedlinePlus

Effect of DFP on neutrophil constitutive apoptosis. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with increasing doses of DFP for 20 hours. Cells were permeabilized with Triton X-100 and then stained with propidium iodide (50 μg/mL). A. Mean fluorescence values are shown for a minimum of 10 000 cells for each condition and are representative of 3 determinations from 13 separate experiments. B. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with or without LPS or DFP (2.5 mM) are represented. Data represent mean ± SD of 15 separate experiments. *P < 0.05. C. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with or without increasing doses of DFP. Data represent mean ± SD of 2 to 8 separate experiments. D. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with LPS or DFP (2.5 mM) from control cells cultured in serum free medium. Results are mean ± SD of 2 separate experiments.
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Figure 2: Effect of DFP on neutrophil constitutive apoptosis. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with increasing doses of DFP for 20 hours. Cells were permeabilized with Triton X-100 and then stained with propidium iodide (50 μg/mL). A. Mean fluorescence values are shown for a minimum of 10 000 cells for each condition and are representative of 3 determinations from 13 separate experiments. B. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with or without LPS or DFP (2.5 mM) are represented. Data represent mean ± SD of 15 separate experiments. *P < 0.05. C. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with or without increasing doses of DFP. Data represent mean ± SD of 2 to 8 separate experiments. D. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with LPS or DFP (2.5 mM) from control cells cultured in serum free medium. Results are mean ± SD of 2 separate experiments.

Mentions: Quiescent neutrophils are constitutively apoptotic; inflammatory stimuli such as LPS inhibit neutrophil apoptosis. Neutrophils were cultured with or without LPS or DFP for 20 hours and apoptosis quantified as the uptake of propidium iodide. LPS (1 μg/mL) significantly inhibited hypodiploid DNA formation compared to control neutrophils. DFP (2.5 mM) caused significantly greater inhibition (Figure 2A &2B), in a dose-dependent manner (Figure 2C). We confirmed that isopropanol, the vehicle in which DFP is dissolved in, did not inhibit neutrophil hypodiploid DNA formation (data not shown). Similar results were obtained when neutrophils were cultured in serum free media (Figure 2D).


Regulation of apoptosis and priming of neutrophil oxidative burst by diisopropyl fluorophosphate.

Tsang JL, Parodo JC, Marshall JC - J Inflamm (Lond) (2010)

Effect of DFP on neutrophil constitutive apoptosis. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with increasing doses of DFP for 20 hours. Cells were permeabilized with Triton X-100 and then stained with propidium iodide (50 μg/mL). A. Mean fluorescence values are shown for a minimum of 10 000 cells for each condition and are representative of 3 determinations from 13 separate experiments. B. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with or without LPS or DFP (2.5 mM) are represented. Data represent mean ± SD of 15 separate experiments. *P < 0.05. C. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with or without increasing doses of DFP. Data represent mean ± SD of 2 to 8 separate experiments. D. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with LPS or DFP (2.5 mM) from control cells cultured in serum free medium. Results are mean ± SD of 2 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913997&req=5

Figure 2: Effect of DFP on neutrophil constitutive apoptosis. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with increasing doses of DFP for 20 hours. Cells were permeabilized with Triton X-100 and then stained with propidium iodide (50 μg/mL). A. Mean fluorescence values are shown for a minimum of 10 000 cells for each condition and are representative of 3 determinations from 13 separate experiments. B. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with or without LPS or DFP (2.5 mM) are represented. Data represent mean ± SD of 15 separate experiments. *P < 0.05. C. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with or without increasing doses of DFP. Data represent mean ± SD of 2 to 8 separate experiments. D. Rate of apoptosis (hypodiploid DNA) of neutrophils treated with LPS or DFP (2.5 mM) from control cells cultured in serum free medium. Results are mean ± SD of 2 separate experiments.
Mentions: Quiescent neutrophils are constitutively apoptotic; inflammatory stimuli such as LPS inhibit neutrophil apoptosis. Neutrophils were cultured with or without LPS or DFP for 20 hours and apoptosis quantified as the uptake of propidium iodide. LPS (1 μg/mL) significantly inhibited hypodiploid DNA formation compared to control neutrophils. DFP (2.5 mM) caused significantly greater inhibition (Figure 2A &2B), in a dose-dependent manner (Figure 2C). We confirmed that isopropanol, the vehicle in which DFP is dissolved in, did not inhibit neutrophil hypodiploid DNA formation (data not shown). Similar results were obtained when neutrophils were cultured in serum free media (Figure 2D).

Bottom Line: Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules.However, DFP enhanced the exteriorization of PS in a dose-dependent manner.We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdepartmental Division of Critical Care, University of Toronto, Toronto, Canada. marshallj@smh.ca.

ABSTRACT

Background: Diisopropyl fluorophosphate (DFP) is a serine protease inhibitor that is widely used as an inhibitor of endogenous proteases in in vitro neutrophil studies. Its effects on neutrophil function are unclear. We sought to determine the biological effects of DFP on human neutrophil apoptosis and oxidative burst.

Methods: We isolated neutrophils from healthy volunteers, incubated them with DFP (2.5 mM), and evaluated neutrophil elastase (NE) activity, neutrophil degranulation, apoptosis as reflected in hypodiploid DNA formation and exteriorization of phosphatidylserine (PS), processing and activity of caspases-3 and -8, oxidative burst activity and hydrogen peroxide release.

Results: Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules. DFP inhibited constitutive neutrophil apoptosis as reflected in DNA fragmentation, and the processing and activity of caspases-3 and -8. DFP also inhibited priming of neutrophils for oxidative burst activity and hydrogen peroxide release. However, DFP enhanced the exteriorization of PS in a dose-dependent manner.

Conclusion: We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity. We further conclude that endogenous proteases play a role in the biology of constitutive neutrophil apoptosis.

No MeSH data available.


Related in: MedlinePlus