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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

PMA induced NET formation: A-M) Cells were treated for 4 h with PMA (A,B: control, C,D: 0.1 nM, E,F 1 nM, G,H 10 nM, I,J 100 nM, K,L 1 μM,) and microscopy performed at a density of 50,000 cells per well in a 96 well plate. Sytox Green cell impermeable dye was added at a concentration of 6 μM. Cells with a typical morphology of diffuse and spread NETs were noted [in this figure 50,000 cells were plated in 1 ml of media in plastic 24 well plates and stimulated as above with 100 nM PMA] (M). Scale bars = 10 microns. A greater abundance of NETs was seen with increases in PMA concentration but abundance was similar in the 10–1000 nM range. N) Fluorescence of extracellular DNA by cell impermeable Sytox Green was measured with an excitation of 485 nm and measured at 530 nm. Background fluorescence was subtracted and the abundance of NETS expressed as fluorescence in arbitrary units. The concentration-dependent response of NET formation plateaued after 10 nM PMA. Data show mean +/− SEM for n = 6 independent experiments.
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Figure 1: PMA induced NET formation: A-M) Cells were treated for 4 h with PMA (A,B: control, C,D: 0.1 nM, E,F 1 nM, G,H 10 nM, I,J 100 nM, K,L 1 μM,) and microscopy performed at a density of 50,000 cells per well in a 96 well plate. Sytox Green cell impermeable dye was added at a concentration of 6 μM. Cells with a typical morphology of diffuse and spread NETs were noted [in this figure 50,000 cells were plated in 1 ml of media in plastic 24 well plates and stimulated as above with 100 nM PMA] (M). Scale bars = 10 microns. A greater abundance of NETs was seen with increases in PMA concentration but abundance was similar in the 10–1000 nM range. N) Fluorescence of extracellular DNA by cell impermeable Sytox Green was measured with an excitation of 485 nm and measured at 530 nm. Background fluorescence was subtracted and the abundance of NETS expressed as fluorescence in arbitrary units. The concentration-dependent response of NET formation plateaued after 10 nM PMA. Data show mean +/− SEM for n = 6 independent experiments.

Mentions: Incubation of human neutrophils with PMA induced dramatic changes in morphology at 4 h after stimulation, resulting in NETs that stained positive with SYTOX green which is impermeable to cells with an intact membrane. The abundance of NETs was almost maximal at 10 nM PMA above which the magnitude of NET formation plateaued (Figure 1A-N). Cells stimulated with PMA demonstrated typical morphology of diffuse and spread NETs (Figure 1M). Measuring the level of total fluorescence with SYTOX green allowed the assessment of total extracellular DNA and thus NET formation (Figure 1N). NET formation determined by microscopy and cell counting (i.e., by expressing the number of areas of extracellular DNA as a percentage of total cell count[14]) strongly correlated with the measurement of NET abundance based on total fluorescence (r2=0.98), data not shown. Therefore total fluorescence was utilised as a reliable screening assay in further experiments to allow a range of inhibitors to be compared, before confirmation with gold-standard microscopic validation. This test was reproducible with an average inter-assay coefficient of variation of 14.3%. As almost maximal NET formation was gained with 10 nM PMA (Figure 1B), this concentration was selected for all further experiments.


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)

PMA induced NET formation: A-M) Cells were treated for 4 h with PMA (A,B: control, C,D: 0.1 nM, E,F 1 nM, G,H 10 nM, I,J 100 nM, K,L 1 μM,) and microscopy performed at a density of 50,000 cells per well in a 96 well plate. Sytox Green cell impermeable dye was added at a concentration of 6 μM. Cells with a typical morphology of diffuse and spread NETs were noted [in this figure 50,000 cells were plated in 1 ml of media in plastic 24 well plates and stimulated as above with 100 nM PMA] (M). Scale bars = 10 microns. A greater abundance of NETs was seen with increases in PMA concentration but abundance was similar in the 10–1000 nM range. N) Fluorescence of extracellular DNA by cell impermeable Sytox Green was measured with an excitation of 485 nm and measured at 530 nm. Background fluorescence was subtracted and the abundance of NETS expressed as fluorescence in arbitrary units. The concentration-dependent response of NET formation plateaued after 10 nM PMA. Data show mean +/− SEM for n = 6 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 1: PMA induced NET formation: A-M) Cells were treated for 4 h with PMA (A,B: control, C,D: 0.1 nM, E,F 1 nM, G,H 10 nM, I,J 100 nM, K,L 1 μM,) and microscopy performed at a density of 50,000 cells per well in a 96 well plate. Sytox Green cell impermeable dye was added at a concentration of 6 μM. Cells with a typical morphology of diffuse and spread NETs were noted [in this figure 50,000 cells were plated in 1 ml of media in plastic 24 well plates and stimulated as above with 100 nM PMA] (M). Scale bars = 10 microns. A greater abundance of NETs was seen with increases in PMA concentration but abundance was similar in the 10–1000 nM range. N) Fluorescence of extracellular DNA by cell impermeable Sytox Green was measured with an excitation of 485 nm and measured at 530 nm. Background fluorescence was subtracted and the abundance of NETS expressed as fluorescence in arbitrary units. The concentration-dependent response of NET formation plateaued after 10 nM PMA. Data show mean +/− SEM for n = 6 independent experiments.
Mentions: Incubation of human neutrophils with PMA induced dramatic changes in morphology at 4 h after stimulation, resulting in NETs that stained positive with SYTOX green which is impermeable to cells with an intact membrane. The abundance of NETs was almost maximal at 10 nM PMA above which the magnitude of NET formation plateaued (Figure 1A-N). Cells stimulated with PMA demonstrated typical morphology of diffuse and spread NETs (Figure 1M). Measuring the level of total fluorescence with SYTOX green allowed the assessment of total extracellular DNA and thus NET formation (Figure 1N). NET formation determined by microscopy and cell counting (i.e., by expressing the number of areas of extracellular DNA as a percentage of total cell count[14]) strongly correlated with the measurement of NET abundance based on total fluorescence (r2=0.98), data not shown. Therefore total fluorescence was utilised as a reliable screening assay in further experiments to allow a range of inhibitors to be compared, before confirmation with gold-standard microscopic validation. This test was reproducible with an average inter-assay coefficient of variation of 14.3%. As almost maximal NET formation was gained with 10 nM PMA (Figure 1B), this concentration was selected for all further experiments.

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