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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 processing of pro-caspases-3 and -8 and caspases-3 and -8 activity. Human neutrophils were incubated alone (Control) or with DFP 2.5 mM for 5 hours. Cells were then lyzed and lysates were separated on 12% SDS-PAGE gel and specific antibodies were used to evaluate the pattern of caspase-8 (A) and caspase-3 (B) processing. Blot is representative of 3 separate experiments. C. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with DFP (2.5 mM) for 5 hours. Cells were then lyzed. Caspase-8 and caspase-3 activities were measured using specific colorimetric and fluorimetric substrates respectively. Caspase-8 (C) activity is represented as absorbance at 405 nm & caspase-3 (D) activity is represented as fluorescence units at excitation wavelength of 360 nm and emission wavelength of 460 nm. Data represent mean ± SD of 4 separate experiments. *P = 0.243 for caspase-8; *P = 0.02 for caspase-3.
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Figure 3: Effect of DFP on processing of pro-caspases-3 and -8 and caspases-3 and -8 activity. Human neutrophils were incubated alone (Control) or with DFP 2.5 mM for 5 hours. Cells were then lyzed and lysates were separated on 12% SDS-PAGE gel and specific antibodies were used to evaluate the pattern of caspase-8 (A) and caspase-3 (B) processing. Blot is representative of 3 separate experiments. C. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with DFP (2.5 mM) for 5 hours. Cells were then lyzed. Caspase-8 and caspase-3 activities were measured using specific colorimetric and fluorimetric substrates respectively. Caspase-8 (C) activity is represented as absorbance at 405 nm & caspase-3 (D) activity is represented as fluorescence units at excitation wavelength of 360 nm and emission wavelength of 460 nm. Data represent mean ± SD of 4 separate experiments. *P = 0.243 for caspase-8; *P = 0.02 for caspase-3.

Mentions: Caspases are synthesized as pro-enzymes that are cleaved at conserved tetra-or pentapeptide amino acid sequences adjacent to aspartic acid residues to form catalytically active enzymes. Spontaneous neutrophil apoptosis can be initiated via either the extrinsic pathway as a consequence of caspase-8 activation following death receptor engagement [26] or the intrinsic pathway as a consequence of loss of mitochondrial transmembrane potential with activation of caspase-9 [27]. Both pathways result in the activation of the downstream effector, caspase-3. Since caspase activation precedes DNA degradation, we studied the effects of DFP on the processing of caspases-3 and -8 at 5 hours. Consistent with an inhibitory effect on apoptosis, pro-caspase-8 was significantly increased (Figure 3A), while the active 12kDa form of caspase-3 was significantly reduced in DFP treated neutrophils (Figure 3B). Moreover DFP inhibited both caspase-8 activity (Figure 3C), and caspase-3 activity (Figure 3D). The inhibition of caspase-3 activity by DFP was significantly greater than that induced by exposure to LPS (p <0.05).


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 processing of pro-caspases-3 and -8 and caspases-3 and -8 activity. Human neutrophils were incubated alone (Control) or with DFP 2.5 mM for 5 hours. Cells were then lyzed and lysates were separated on 12% SDS-PAGE gel and specific antibodies were used to evaluate the pattern of caspase-8 (A) and caspase-3 (B) processing. Blot is representative of 3 separate experiments. C. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with DFP (2.5 mM) for 5 hours. Cells were then lyzed. Caspase-8 and caspase-3 activities were measured using specific colorimetric and fluorimetric substrates respectively. Caspase-8 (C) activity is represented as absorbance at 405 nm & caspase-3 (D) activity is represented as fluorescence units at excitation wavelength of 360 nm and emission wavelength of 460 nm. Data represent mean ± SD of 4 separate experiments. *P = 0.243 for caspase-8; *P = 0.02 for caspase-3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effect of DFP on processing of pro-caspases-3 and -8 and caspases-3 and -8 activity. Human neutrophils were incubated alone (Control) or with DFP 2.5 mM for 5 hours. Cells were then lyzed and lysates were separated on 12% SDS-PAGE gel and specific antibodies were used to evaluate the pattern of caspase-8 (A) and caspase-3 (B) processing. Blot is representative of 3 separate experiments. C. Human neutrophils were incubated alone (Control), with LPS (1 μg/mL) or with DFP (2.5 mM) for 5 hours. Cells were then lyzed. Caspase-8 and caspase-3 activities were measured using specific colorimetric and fluorimetric substrates respectively. Caspase-8 (C) activity is represented as absorbance at 405 nm & caspase-3 (D) activity is represented as fluorescence units at excitation wavelength of 360 nm and emission wavelength of 460 nm. Data represent mean ± SD of 4 separate experiments. *P = 0.243 for caspase-8; *P = 0.02 for caspase-3.
Mentions: Caspases are synthesized as pro-enzymes that are cleaved at conserved tetra-or pentapeptide amino acid sequences adjacent to aspartic acid residues to form catalytically active enzymes. Spontaneous neutrophil apoptosis can be initiated via either the extrinsic pathway as a consequence of caspase-8 activation following death receptor engagement [26] or the intrinsic pathway as a consequence of loss of mitochondrial transmembrane potential with activation of caspase-9 [27]. Both pathways result in the activation of the downstream effector, caspase-3. Since caspase activation precedes DNA degradation, we studied the effects of DFP on the processing of caspases-3 and -8 at 5 hours. Consistent with an inhibitory effect on apoptosis, pro-caspase-8 was significantly increased (Figure 3A), while the active 12kDa form of caspase-3 was significantly reduced in DFP treated neutrophils (Figure 3B). Moreover DFP inhibited both caspase-8 activity (Figure 3C), and caspase-3 activity (Figure 3D). The inhibition of caspase-3 activity by DFP was significantly greater than that induced by exposure to LPS (p <0.05).

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