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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 exteriorization of phosphatidylserine quantified as the uptake of Annexin V. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with increasing doses of DFP for 5 hours. Cells were centrifuged and incubated with FITC-conjugated Annexin. Phosphatidylserine exteriorization was detected as mean channel fluorescence at an excitation wavelength of 388 nm and emission wavelength of 520 nm. A. Phosphatidylserine exteriorization of neutrophils treated with or without LPS or DFP (2.5 mM). Data represent mean ± SD of 4 separate experiments. *P = 0.349 versus controls, p < 0.001 versus LPS. B. Phosphatidylserine exteriorization of neutrophils treated with or without various doses of DFP. Data represent mean ± SD of 2 to 3 separate experiments.
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Figure 4: Effect of DFP on exteriorization of phosphatidylserine quantified as the uptake of Annexin V. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with increasing doses of DFP for 5 hours. Cells were centrifuged and incubated with FITC-conjugated Annexin. Phosphatidylserine exteriorization was detected as mean channel fluorescence at an excitation wavelength of 388 nm and emission wavelength of 520 nm. A. Phosphatidylserine exteriorization of neutrophils treated with or without LPS or DFP (2.5 mM). Data represent mean ± SD of 4 separate experiments. *P = 0.349 versus controls, p < 0.001 versus LPS. B. Phosphatidylserine exteriorization of neutrophils treated with or without various doses of DFP. Data represent mean ± SD of 2 to 3 separate experiments.

Mentions: We next sought to determine the effects of DFP on a separate early event in apoptosis, the exteriorization of phosphatidylserine (PS) on the cell membrane. Contrary to the effects of DFP on hypodiploid DNA formation, culture of neutrophils with DFP (2.5 mM) for 5 hours significantly increased the amount of exteriorized PS as demonstrated by increased binding of Annexin V (Figure 4A). Increased exteriorization of PS was dose-dependent (Figure 4B). In contrast, culture of neutrophils with LPS (1 μg/mL) for 5 hours suppressed the exteriorization of PS (Figure 4A). Thus DFP exerts differential effects on early and late events in the progression of apoptosis. Isopropanol had no effect on the exteriorization of phosphatidylserine (data not shown).


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 exteriorization of phosphatidylserine quantified as the uptake of Annexin V. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with increasing doses of DFP for 5 hours. Cells were centrifuged and incubated with FITC-conjugated Annexin. Phosphatidylserine exteriorization was detected as mean channel fluorescence at an excitation wavelength of 388 nm and emission wavelength of 520 nm. A. Phosphatidylserine exteriorization of neutrophils treated with or without LPS or DFP (2.5 mM). Data represent mean ± SD of 4 separate experiments. *P = 0.349 versus controls, p < 0.001 versus LPS. B. Phosphatidylserine exteriorization of neutrophils treated with or without various doses of DFP. Data represent mean ± SD of 2 to 3 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effect of DFP on exteriorization of phosphatidylserine quantified as the uptake of Annexin V. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with increasing doses of DFP for 5 hours. Cells were centrifuged and incubated with FITC-conjugated Annexin. Phosphatidylserine exteriorization was detected as mean channel fluorescence at an excitation wavelength of 388 nm and emission wavelength of 520 nm. A. Phosphatidylserine exteriorization of neutrophils treated with or without LPS or DFP (2.5 mM). Data represent mean ± SD of 4 separate experiments. *P = 0.349 versus controls, p < 0.001 versus LPS. B. Phosphatidylserine exteriorization of neutrophils treated with or without various doses of DFP. Data represent mean ± SD of 2 to 3 separate experiments.
Mentions: We next sought to determine the effects of DFP on a separate early event in apoptosis, the exteriorization of phosphatidylserine (PS) on the cell membrane. Contrary to the effects of DFP on hypodiploid DNA formation, culture of neutrophils with DFP (2.5 mM) for 5 hours significantly increased the amount of exteriorized PS as demonstrated by increased binding of Annexin V (Figure 4A). Increased exteriorization of PS was dose-dependent (Figure 4B). In contrast, culture of neutrophils with LPS (1 μg/mL) for 5 hours suppressed the exteriorization of PS (Figure 4A). Thus DFP exerts differential effects on early and late events in the progression of apoptosis. Isopropanol had no effect on the exteriorization of phosphatidylserine (data not shown).

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