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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

Effect of DPI and Ro-31-8220 on NET formation: Cells were plated as described and either pre-treated with DPI or Ro-31-8220 at increasing concentrations, or control media, before stimulating with PMA at 10 nM for 4 h. NET abundance was measured by total fluorescence from Sytox Green cell impermeable dye. A) Pre-treatment with Ro-31-8220 completely abrogated NET formation at 100 nM and 1 μM. Data show mean +/− SEM for n=4 independent experiments. *** indicates p<0.001. B) Pre-treatment with DPI significantly decreased NET formation at 10 nM, and completely abrogated NET formation at 100 nM and 1 μM. Data show mean +/− SEM for n=4 independent experiments. *** indicates p<0.001.
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Figure 2: Effect of DPI and Ro-31-8220 on NET formation: Cells were plated as described and either pre-treated with DPI or Ro-31-8220 at increasing concentrations, or control media, before stimulating with PMA at 10 nM for 4 h. NET abundance was measured by total fluorescence from Sytox Green cell impermeable dye. A) Pre-treatment with Ro-31-8220 completely abrogated NET formation at 100 nM and 1 μM. Data show mean +/− SEM for n=4 independent experiments. *** indicates p<0.001. B) Pre-treatment with DPI significantly decreased NET formation at 10 nM, and completely abrogated NET formation at 100 nM and 1 μM. Data show mean +/− SEM for n=4 independent experiments. *** indicates p<0.001.

Mentions: In order to determine whether NET formation was dependent on the activation of PKC and NADPH oxidase, cells were preincubated with increasing concentrations of the specific but isozyme non-selective PKC inhibitor, Ro-31-8220 or the NADPH inhibitor diphenyloidonium (DPI) for 30 min and then treated with 10 nM PMA. Both Ro-31-8220 and DPI, completely inhibited PMA induced NET formation (Figure 2A and2B) measured by SYTOX green fluorescence and confirmed by microscopy. These data confirm the key role of NADPH oxidase and demonstrate that the NET-forming activity of PMA is critically dependent upon PKC pathways upstream of NADPH oxidase.


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)

Effect of DPI and Ro-31-8220 on NET formation: Cells were plated as described and either pre-treated with DPI or Ro-31-8220 at increasing concentrations, or control media, before stimulating with PMA at 10 nM for 4 h. NET abundance was measured by total fluorescence from Sytox Green cell impermeable dye. A) Pre-treatment with Ro-31-8220 completely abrogated NET formation at 100 nM and 1 μM. Data show mean +/− SEM for n=4 independent experiments. *** indicates p<0.001. B) Pre-treatment with DPI significantly decreased NET formation at 10 nM, and completely abrogated NET formation at 100 nM and 1 μM. Data show mean +/− SEM for n=4 independent experiments. *** indicates p<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Effect of DPI and Ro-31-8220 on NET formation: Cells were plated as described and either pre-treated with DPI or Ro-31-8220 at increasing concentrations, or control media, before stimulating with PMA at 10 nM for 4 h. NET abundance was measured by total fluorescence from Sytox Green cell impermeable dye. A) Pre-treatment with Ro-31-8220 completely abrogated NET formation at 100 nM and 1 μM. Data show mean +/− SEM for n=4 independent experiments. *** indicates p<0.001. B) Pre-treatment with DPI significantly decreased NET formation at 10 nM, and completely abrogated NET formation at 100 nM and 1 μM. Data show mean +/− SEM for n=4 independent experiments. *** indicates p<0.001.
Mentions: In order to determine whether NET formation was dependent on the activation of PKC and NADPH oxidase, cells were preincubated with increasing concentrations of the specific but isozyme non-selective PKC inhibitor, Ro-31-8220 or the NADPH inhibitor diphenyloidonium (DPI) for 30 min and then treated with 10 nM PMA. Both Ro-31-8220 and DPI, completely inhibited PMA induced NET formation (Figure 2A and2B) measured by SYTOX green fluorescence and confirmed by microscopy. These data confirm the key role of NADPH oxidase and demonstrate that the NET-forming activity of PMA is critically dependent upon PKC pathways upstream of NADPH oxidase.

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