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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 PKC β on NET formation in response to PMA and DAG analogue OAG: Cells were plated as described and pre-treated with increasing concentrations of LY333531 (PKCβ inhibitor) before treatment with either 10 nM PMA or 20 μM 1-oleoyl-2-acetyl-sn-glycerol (OAG) for 4 h. A) Pre-Treatment with increasing concentrations of LY333531 decreases NET formation in response to 10 nM PMA. Data show mean +/− SEM for n=6 independent experiments for PMA stimulated cells and n=3 for OAG stimulated cells. *indicates p<0.05, ***p<0.001. B). Pre-treatment with increasing concentrations of LY333531 completely abrogated NET formation in response to 20 μM OAG. Data show mean +/− SEM for n=3 independent experiments. *** indicates p<0.001. C-J). Pre-treatment with 1 μM LY333531 completely abrogated NET formation in response to PMA and OAG, images representative of n=3 independent experiments (C,D Cells treated with 10 nM PMA. E,F cells pre-treated with 1 μM LY333531 for 30 min then 10 nM PMA. G,H cells treated with 20 μM OAG. I,G Cells pre-treated with 1 μM LY333531 for 30 min then 20 μM OAG. K,L Higher magnification images of effects of treatment with 100 nM LY333531 (L) prior to 10 nM PMA. Fluorescent and phase images combined. There is an absence of spreading NET material in the LY333531 treated cells although some diffuse NET formation is still present at this concentration of inhibitor. Scale bars = 10 microns.
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Figure 4: Effect of PKC β on NET formation in response to PMA and DAG analogue OAG: Cells were plated as described and pre-treated with increasing concentrations of LY333531 (PKCβ inhibitor) before treatment with either 10 nM PMA or 20 μM 1-oleoyl-2-acetyl-sn-glycerol (OAG) for 4 h. A) Pre-Treatment with increasing concentrations of LY333531 decreases NET formation in response to 10 nM PMA. Data show mean +/− SEM for n=6 independent experiments for PMA stimulated cells and n=3 for OAG stimulated cells. *indicates p<0.05, ***p<0.001. B). Pre-treatment with increasing concentrations of LY333531 completely abrogated NET formation in response to 20 μM OAG. Data show mean +/− SEM for n=3 independent experiments. *** indicates p<0.001. C-J). Pre-treatment with 1 μM LY333531 completely abrogated NET formation in response to PMA and OAG, images representative of n=3 independent experiments (C,D Cells treated with 10 nM PMA. E,F cells pre-treated with 1 μM LY333531 for 30 min then 10 nM PMA. G,H cells treated with 20 μM OAG. I,G Cells pre-treated with 1 μM LY333531 for 30 min then 20 μM OAG. K,L Higher magnification images of effects of treatment with 100 nM LY333531 (L) prior to 10 nM PMA. Fluorescent and phase images combined. There is an absence of spreading NET material in the LY333531 treated cells although some diffuse NET formation is still present at this concentration of inhibitor. Scale bars = 10 microns.

Mentions: Gö 6976 is an inhibitor of both PKCα and β, thus, in order to separate the roles of these two isozymes, a specific PKCβ inhibitor (LY333531) was employed (Figure 4). LY333531 significantly inhibited NET formation by PMA at a concentration of 100 nM (p<0.05) and 1 μM (p<0.01) (Figure 4A). In addition, 1-oleoyl-2-acetyl-sn-glycerol (OAG; a DAG analogue and activator of conventional PKC isoforms) stimulated NET formation in a manner similar to that of PMA (albeit at higher concentrations) and was also significantly inhibited by 1 μM LY333531 (p<0.01) (Figure 4B). These data were confirmed by microscopy and delineate a central role for PKC β in PMA and OAG-induced NET formation (Figure 4C-J). Interestingly as well as knocking down total NET production LY333531 completely abrogated the presence of “spreading NETs” at the 100 nm concentration but some diffuse NET formation was still observed (Figure 4K,L).


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 PKC β on NET formation in response to PMA and DAG analogue OAG: Cells were plated as described and pre-treated with increasing concentrations of LY333531 (PKCβ inhibitor) before treatment with either 10 nM PMA or 20 μM 1-oleoyl-2-acetyl-sn-glycerol (OAG) for 4 h. A) Pre-Treatment with increasing concentrations of LY333531 decreases NET formation in response to 10 nM PMA. Data show mean +/− SEM for n=6 independent experiments for PMA stimulated cells and n=3 for OAG stimulated cells. *indicates p<0.05, ***p<0.001. B). Pre-treatment with increasing concentrations of LY333531 completely abrogated NET formation in response to 20 μM OAG. Data show mean +/− SEM for n=3 independent experiments. *** indicates p<0.001. C-J). Pre-treatment with 1 μM LY333531 completely abrogated NET formation in response to PMA and OAG, images representative of n=3 independent experiments (C,D Cells treated with 10 nM PMA. E,F cells pre-treated with 1 μM LY333531 for 30 min then 10 nM PMA. G,H cells treated with 20 μM OAG. I,G Cells pre-treated with 1 μM LY333531 for 30 min then 20 μM OAG. K,L Higher magnification images of effects of treatment with 100 nM LY333531 (L) prior to 10 nM PMA. Fluorescent and phase images combined. There is an absence of spreading NET material in the LY333531 treated cells although some diffuse NET formation is still present at this concentration of inhibitor. Scale bars = 10 microns.
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Related In: Results  -  Collection

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Figure 4: Effect of PKC β on NET formation in response to PMA and DAG analogue OAG: Cells were plated as described and pre-treated with increasing concentrations of LY333531 (PKCβ inhibitor) before treatment with either 10 nM PMA or 20 μM 1-oleoyl-2-acetyl-sn-glycerol (OAG) for 4 h. A) Pre-Treatment with increasing concentrations of LY333531 decreases NET formation in response to 10 nM PMA. Data show mean +/− SEM for n=6 independent experiments for PMA stimulated cells and n=3 for OAG stimulated cells. *indicates p<0.05, ***p<0.001. B). Pre-treatment with increasing concentrations of LY333531 completely abrogated NET formation in response to 20 μM OAG. Data show mean +/− SEM for n=3 independent experiments. *** indicates p<0.001. C-J). Pre-treatment with 1 μM LY333531 completely abrogated NET formation in response to PMA and OAG, images representative of n=3 independent experiments (C,D Cells treated with 10 nM PMA. E,F cells pre-treated with 1 μM LY333531 for 30 min then 10 nM PMA. G,H cells treated with 20 μM OAG. I,G Cells pre-treated with 1 μM LY333531 for 30 min then 20 μM OAG. K,L Higher magnification images of effects of treatment with 100 nM LY333531 (L) prior to 10 nM PMA. Fluorescent and phase images combined. There is an absence of spreading NET material in the LY333531 treated cells although some diffuse NET formation is still present at this concentration of inhibitor. Scale bars = 10 microns.
Mentions: Gö 6976 is an inhibitor of both PKCα and β, thus, in order to separate the roles of these two isozymes, a specific PKCβ inhibitor (LY333531) was employed (Figure 4). LY333531 significantly inhibited NET formation by PMA at a concentration of 100 nM (p<0.05) and 1 μM (p<0.01) (Figure 4A). In addition, 1-oleoyl-2-acetyl-sn-glycerol (OAG; a DAG analogue and activator of conventional PKC isoforms) stimulated NET formation in a manner similar to that of PMA (albeit at higher concentrations) and was also significantly inhibited by 1 μM LY333531 (p<0.01) (Figure 4B). These data were confirmed by microscopy and delineate a central role for PKC β in PMA and OAG-induced NET formation (Figure 4C-J). Interestingly as well as knocking down total NET production LY333531 completely abrogated the presence of “spreading NETs” at the 100 nm concentration but some diffuse NET formation was still observed (Figure 4K,L).

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