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Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps.

Gray RD, Lucas CD, Mackellar A, Li F, Hiersemenzel K, Haslett C, Davidson DJ, Rossi AG - J Inflamm (Lond) (2013)

Bottom Line: Inhibition of novel and atypical PKC had no effect.Conventional PKCs have a prominent role in NET formation.Furthermore PKCβ is the major isoform implicated in NET formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh Medical School, 47 Little France Crescent, Edinburgh, Scotland, UK. r.d.gray@ed.ac.uk.

ABSTRACT

Background: Activation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation.

Methods: Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood.

Results: Pan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCβ inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG (conventional PKC activator) (p<0.001).

Conclusions: Conventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation.

No MeSH data available.


Related in: MedlinePlus

The downstream effect of PKC β inhibition on oxidative burst. Cells were pretreated with LY333531 (Ly) at increasing concentrations and NADPH oxidase activity measured by DHR fluorescence on flow-cytometry. DPI and Ro-31-8220 (Ro) were used as positive controls. LY333531 reduced NADPH oxidase activity at both 100 nM (p<0.01) and 1 μM (p<0.001) consistent with the concentrations of inhibitor required to reduce NET formation. # indicates p<0.05, ## indicates p<0.01, and ### indicates p<0.001 vs. untreated control. ** indicates p<0.01 and *** indicates p<0.001 vs. PMA treated cells.
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Figure 5: The downstream effect of PKC β inhibition on oxidative burst. Cells were pretreated with LY333531 (Ly) at increasing concentrations and NADPH oxidase activity measured by DHR fluorescence on flow-cytometry. DPI and Ro-31-8220 (Ro) were used as positive controls. LY333531 reduced NADPH oxidase activity at both 100 nM (p<0.01) and 1 μM (p<0.001) consistent with the concentrations of inhibitor required to reduce NET formation. # indicates p<0.05, ## indicates p<0.01, and ### indicates p<0.001 vs. untreated control. ** indicates p<0.01 and *** indicates p<0.001 vs. PMA treated cells.

Mentions: To elucidate the downstream effects of PKCβ inhibition we assessed the effects of LY333531 on oxidative burst as measured by DHR fluorescence on flow cytometry. LY333531 reduced NADPH oxidative burst at the same concentrations required to reduce NET formation, with a partial but significant knock down of activity at 100 nM (p<0.01) and a complete knockdown at 1 μM (p<0.001) in comparison to the positive controls of DPI and Ro-31-8220 (Figure 5).


Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps.

Gray RD, Lucas CD, Mackellar A, Li F, Hiersemenzel K, Haslett C, Davidson DJ, Rossi AG - J Inflamm (Lond) (2013)

The downstream effect of PKC β inhibition on oxidative burst. Cells were pretreated with LY333531 (Ly) at increasing concentrations and NADPH oxidase activity measured by DHR fluorescence on flow-cytometry. DPI and Ro-31-8220 (Ro) were used as positive controls. LY333531 reduced NADPH oxidase activity at both 100 nM (p<0.01) and 1 μM (p<0.001) consistent with the concentrations of inhibitor required to reduce NET formation. # indicates p<0.05, ## indicates p<0.01, and ### indicates p<0.001 vs. untreated control. ** indicates p<0.01 and *** indicates p<0.001 vs. PMA treated cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643828&req=5

Figure 5: The downstream effect of PKC β inhibition on oxidative burst. Cells were pretreated with LY333531 (Ly) at increasing concentrations and NADPH oxidase activity measured by DHR fluorescence on flow-cytometry. DPI and Ro-31-8220 (Ro) were used as positive controls. LY333531 reduced NADPH oxidase activity at both 100 nM (p<0.01) and 1 μM (p<0.001) consistent with the concentrations of inhibitor required to reduce NET formation. # indicates p<0.05, ## indicates p<0.01, and ### indicates p<0.001 vs. untreated control. ** indicates p<0.01 and *** indicates p<0.001 vs. PMA treated cells.
Mentions: To elucidate the downstream effects of PKCβ inhibition we assessed the effects of LY333531 on oxidative burst as measured by DHR fluorescence on flow cytometry. LY333531 reduced NADPH oxidative burst at the same concentrations required to reduce NET formation, with a partial but significant knock down of activity at 100 nM (p<0.01) and a complete knockdown at 1 μM (p<0.001) in comparison to the positive controls of DPI and Ro-31-8220 (Figure 5).

Bottom Line: Inhibition of novel and atypical PKC had no effect.Conventional PKCs have a prominent role in NET formation.Furthermore PKCβ is the major isoform implicated in NET formation.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Inflammation Research, The Queen's Medical Research Institute, University of Edinburgh Medical School, 47 Little France Crescent, Edinburgh, Scotland, UK. r.d.gray@ed.ac.uk.

ABSTRACT

Background: Activation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation.

Methods: Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood.

Results: Pan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCβ inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG (conventional PKC activator) (p<0.001).

Conclusions: Conventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation.

No MeSH data available.


Related in: MedlinePlus