- Short Report
- Open Access
Activation of conventional protein kinase C (PKC) is critical in the generation of human neutrophil extracellular traps
- Robert D Gray1Email author,
- Christopher D Lucas1,
- Annie MacKellar1,
- Feng Li1,
- Katia Hiersemenzel1,
- Chris Haslett1,
- Donald J Davidson†1 and
- Adriano G Rossi†1
© Gray et al.; licensee BioMed Central Ltd. 2013
Received: 4 September 2012
Accepted: 22 February 2013
Published: 21 March 2013
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.
Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood.
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).
Conventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation.
Neutrophil granulocytes are key cells of the innate immune system with a primary function of killing invading microorganisms such as bacteria, fungi and parasites to prevent pathogenic spread and invasion[1, 2]. Once identified, neutrophils phagocytose and destroy microbes inside the phagolysosome by localised disgorgement of granule contents and the generation of reactive oxygen species (ROS). Engulfment of the microorganism allows killing to take place in a confined area within the cell and not in the extracellular space. Neutrophils may also liberate granule contents and ROS into the surrounding extracellular space to destroy nearby foreign pathogens. Dysregulation of these processes may cause histotoxic damage surrounding host cells. More recently a further extracellular killing mechanism available to neutrophils has been described known as neutrophil extracellular trap (NET) formation[3, 4]. NETs are formed by the mixing of cytoplasmic contents with nuclear histones and DNA to form a network which is propelled to the exterior of the cell. Microbes are caught in this mesh and killed by the neutrophil proteins and histones contained in the NETs. This process of NET formation leads to a form of cell death, NETosis, that has been characterised as being different from either apoptosis or necrosis.
NET formation is known to be stimulated by specific cytokines (e.g., interleukin 8 (IL-8)), bacterial products (e.g., lipopolysaccharide (LPS)) and importantly by clinically relevant pathogens such as Shigella flexneri, Staphylococcus aureus, Salmonella thyphimurium, Streptococcus pneumoniae and the fungus Candida albicans. Stimulation and activation of neutrophils with the diacylglycerol (DAG) mimetic phorbol 12-myristate 13-acetate (PMA) also results in the production of NETs and has given important clues as to the possible mechanism involved in the formation of such structures. It is clear that NET formation following PMA stimulation is dependent on ROS production (via the NADPH oxidase system) and this is likely to follow the activation of protein kinase C (PKC) as well as other pathways such as raf-MEK-ERK.
The PKC isozyme family is comprised of conventional, novel and atypical isoforms. There are at least four conventional isozymes: PKCα, PKCβI, PKCβII and PKCγ. The novel isozyme group has four subtypes: PKCδ, PKCε, PKCη and PKCθ. The third group, atypical isozymes, consists of PKCζ and PKCι. PMA stimulates conventional (α, βI, βII, γ) and novel (δ, ε, η, θ) PKC by mimicking the activating ligand DAG. PKC isoforms of all classifications have been reported in neutrophils from healthy donors. Given that PMA activation triggers NET formation, we hypothesised that specific isoform(s) of PKC are a key modulator of the NET formation pathway. To address this hypothesis we evaluated a panel of PKC inhibitors on NET formation.
Material and methods
Dihydrorhodamine (DHR), dimethyl sulfoxide (DMSO), diphenyliodonium (DPI), Phorbol 12-myristate 13-acetate (PMA), Ro-31-8220, PKCζ pseudosubstrate myristoyl trifluoroacetate (PKCζ inhibitor) and SYTOX green were purchased from Sigma-Aldrich (Dorset, UK); Rottlerin, Gö 6976 and LY333531 were from Calbiochem (Merck) (Darmstadt, Germany).
Isolation of human neutrophils
Peripheral blood neutrophils were isolated from healthy human volunteers according to Lothian Research Ethics Committee approvals #08/S1103/38 via dextran sedimentation and Percoll™ discontinuous gradients as described[11, 12]. Informed written consent was obtained from all subjects. Purity of the neutrophils was assessed by examination of cytocentrifuge preparations and was greater than 95%.
Assessment of NET formation
Neutrophils (5×104 cells/well) in HBSS containing Ca2+, Mg2+ and Hepes (20 mM) were aliquoted (180 μl) into 96 well plates and left to settle for 30 min at 37°C. The inhibitors Ro-31-8220, DPI, rottlerin, PKCζ inhibitor, Gö 6976 and PKCβ inhibitor were added at appropriate concentrations to wells in duplicates and incubated for 30 min before adding PMA. The final volume in each well was 200 μl. Plates were incubated for 4 h and then SYTOX green (6 μM final concentration), a cell-impermeable nucleic acid stain, with an excitation/emission maxima of 504/523 nm to give a green fluorescent light, was added and NET formation was observed by measuring mean fluorescence in 96 well plates. In some experiments 1-oleoyl-2-acetyl-sn-glycerol (OAG) was used to stimulate cells in place of PMA. Results were evaluated by measuring the mean fluorescence in 96 well plates after the subtraction of background fluorescence. Cells were also visualised by fluorescent microscopy carried out on a Zeiss Axiovert S100 fluorescent microscope (Carl Zeiss, Germany) and an Evos fl inverted microscope (AMG, Bothwell, WA).
Data were assessed by one way ANOVA followed by a post-hoc Dunnett’s test. The data were expressed as mean ± standard error of the mean (SEM), and values of p < 0.05 were considered statistically significant. All statistics were performed using GraphPad Prism 5 software (GraphPad, CA, USA).
PMA induced NET formation
NET formation is PKC and NADPH oxidase dependent
Specific PKC isoforms regulated NET formation
PKC β is Primarily Implicated in NET formation in Response to PMA and OAG
PKCβ inhibition has downstream effects on oxidative burst
Assessment of downstream effects of PKCβ inhibition with specific inhibitors
ROS generation was assessed by dihydrorhodamine (DHR) fluorescence as described previously. Neutrophils were resuspended in HBSS with cations and loaded with DHR (2 M; Invitrogen, Carlsbad, CA, USA) for 10 min. Cells were then incubated with or without PKCβ inhibitor (at 10, 100 and 1000 nM) or the positive controls Ro-31-8220 or DPI at 1μM on a shaking heat block for 30 min before stimulation with PMA 10 nM for a further 15 min. DHR fluorescence was analyzed by flow cytometry (FL-1).
The results clearly show that NET formation induced by PMA is PKC and NADPH oxidase dependent. NET formation was blocked by both Pan-PKC inhibition and conventional-PKC inhibition. Furthermore, a specific PKCβ inhibitor (LY333531) also blocked NET formation. LY333531 has high selectivity for PKCβ over other conventional isoforms (IC50 of around 5 nM) with a 60 fold selectivity for PKCβ over PKCα. At higher concentrations specific inhibitors may have non-selective effects on other PKC isoforms. The IC50 of LY333531 for PKCα is around 300 nM, suggesting that the majority of the effect of this compound at the concentrations utilised in our study is via the inhibition of PKCβ and not PKCα; this is evidenced by the significant reduction in NET formation with 100 nM LY333531. The intracellular concentration of LY333531 within the neutrophil following incubation is unknown but it is unlikely to be fully absorbed and as such again we would suggest the effects are due to inhibition of PKCβ. Previous work has demonstrated that PKCβ accounts for 50% of the neutrophil response to PMA further underlining the likely predominant role of PKCβ in NET production.
Oxidative burst and the generation of reactive oxygen species including superoxide anions (02-) and nitric oxide (NO) are fundamental responses of the neutrophil to inflammatory stimuli and pathogens. NET formation is dependent on NADPH oxidase activation and consequently on the generation of 02- which can be blocked by DPI. DPI inhibits NADPH oxidase by binding to specific subunits in the enzyme complex and preventing electron flow and 02- production. The main component of NADPH oxidase is the flavocytochrome b558, a dimer of p22phox and gp91phox, which is an active transporter of electrons across the membrane. Coupled to these are proteins p40phox, p47phox, p67phox and p21rac which are crucial to electron translocation. These proteins assemble when activated to produce 02- which are then spontaneously converted to H2O2. Interestingly, p47phox has to be phosphorylated to acquire a conformational rearrangement to expose the domains that are important for the NADPH oxidase function, and this phosphorylation is mediated by PKC. This is consistent with our findings that PKC is involved in PMA induced NET formation and furthermore that PKCβ is the isoform crucially involved. This is further underlined by the finding that oxidative burst is reduced by concentrations of LY333531 that reduce NET formation.
The beneficial anti-microbial effects of NET formation have been described in several studies[4, 20–25]. Indeed, this is perhaps most pertinently displayed in restoration of NADPH oxidase function in chronic granulomatous disease by gene therapy leading to an increased resistance to fungal infection and clinical improvement secondary to the restoration of the ability to form NETs. Several studies however have demonstrated a pro-inflammatory potential of NETs in a diverse range of diseases including systemic lupus erythematosus[26–28], cystic fibrosis[1, 29, 30] and psoriasis. Therefore the modulation of NET production may be a viable anti-inflammatory target. Inhibition of PKC activity represents one such target as PKC inhibitors have been in development for many years as potential anti-cancer therapies, many of which are orally bioavailable. Furthermore the relative redundancy in PKC function due to multiple isoforms may allow the targeting of specific PKCs in specific cell types at specific organ sites. PKCβ knock out in a murine model has been demonstrated to modulate ischemia reperfusion injury in vivo, however these mice may also be immunodeficient and thus caution must be exercised in any strategy to specifically target PKC. Extracellular traps from both neutrophils and mast cells have been demonstrated in psoriatic skin lesions and from purified neutrophils from psoriasis patients in association with IL-17 and MPO, directly implicating extracellular traps in the pathogenesis of disease. A previous study of a PKC inhibitor AEB071 with specificity for PKC α, β, and θ in psoriasis demonstrated not only in-vitro effects on T cell proliferation and cytokine production but also a clinical improvement in psoriatic lesions in treated patients. We may infer that some of this effect may be due to a direct effect of PKC inhibition on NET formation and thus inflammation in the skin lesions of these patients. Further studies will of course be required to support this hypothesis.
In summary, NET formation in response to PMA and DAG analogues is dependent on PKC activation. Furthermore, we demonstrate that conventional PKC and in particular PKCβ is the predominant isoform responsible for NET formation under these conditions. Although NETs have been demonstrated to entrap and kill various microorganisms there is burgeoning evidence implicating a role for these structures in inflammatory disease and potential modulation of NET production (by PKC inhibition) may offer a novel anti-inflammatory strategy.
RDG is a Wellcome Trust Fellow (093767). DD is an MRC Senior Research Fellow (G1002046). This work was also funded by the Wellcome Trust (WT094415; CL) and the MRC (G0601481; AGR and CH).
- Papayannopoulos V, Staab D, Zychlinsky A: Neutrophil elastase enhances sputum solubilization in cystic fibrosis patients receiving DNase therapy. PLoS One. 2011, 6: e28526-10.1371/journal.pone.0028526.PubMedPubMed CentralView ArticleGoogle Scholar
- Nathan C: Neutrophils and immunity: challenges and opportunities. Nat Rev Immunol. 2006, 6: 173-182. 10.1038/nri1785.PubMedView ArticleGoogle Scholar
- Brinkmann V, Zychlinsky A: Beneficial suicide: why neutrophils die to make NETs. Nat Rev Microbiol. 2007, 5: 577-582. 10.1038/nrmicro1710.PubMedView ArticleGoogle Scholar
- Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, Weinrauch Y, Zychlinsky A: Neutrophil extracellular traps kill bacteria. Science. 2004, 303: 1532-1535. 10.1126/science.1092385.PubMedView ArticleGoogle Scholar
- Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, Weinrauch Y, Brinkmann V, Zychlinsky A: Novel cell death program leads to neutrophil extracellular traps. J Cell Biol. 2007, 176: 231-241. 10.1083/jcb.200606027.PubMedPubMed CentralView ArticleGoogle Scholar
- Medina E: Neutrophil extracellular traps: a strategic tactic to defeat pathogens with potential consequences for the host. J Innate Immun. 2009, 1: 176-180. 10.1159/000203699.PubMedView ArticleGoogle Scholar
- Hakkim A, Fuchs TA, Martinez NE, Hess S, Prinz H, Zychlinsky A, Waldmann H: Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation. Nat Chem Biol. 2011, 7: 75-77.PubMedView ArticleGoogle Scholar
- Way KJ, Chou E, King GL: Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol Sci. 2000, 21: 181-187. 10.1016/S0165-6147(00)01468-1.PubMedView ArticleGoogle Scholar
- Roffey J, Rosse C, Linch M, Hibbert A, McDonald NQ, Parker PJ: Protein kinase C intervention: the state of play. Curr Opin Cell Biol. 2009, 21: 268-279. 10.1016/j.ceb.2009.01.019.PubMedView ArticleGoogle Scholar
- Balasubramanian N, Advani SH, Zingde SM: Protein kinase C isoforms in normal and leukemic neutrophils: altered levels in leukemic neutrophils and changes during myeloid maturation in chronic myeloid leukemia. Leuk Res. 2002, 26: 67-81. 10.1016/S0145-2126(01)00098-4.PubMedView ArticleGoogle Scholar
- Haslett C, Guthrie LA, Kopaniak MM, Johnston RB, Henson PM: Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol. 1985, 119: 101-110.PubMedPubMed CentralGoogle Scholar
- Rossi AG, Sawatzky DA, Walker A, Ward C, Sheldrake TA, Riley NA, Caldicott A, Martinez-Losa M, Walker TR, Duffin R: Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis. Nat Med. 2006, 12: 1056-1064. 10.1038/nm1468.PubMedView ArticleGoogle Scholar
- Lucas CD, Allen KC, Dorward DA, Hoodless LJ, Melrose LA, Marwick JA, Tucker CS, Haslett C, Duffin R, Rossi AG: Flavones induce neutrophil apoptosis by down-regulation of Mcl-1 via a proteasomal-dependent pathway. FASEB J. 2013, 27 (3): 1084-1094. 10.1096/fj.12-218990.PubMedPubMed CentralView ArticleGoogle Scholar
- Metzler KD, Fuchs TA, Nauseef WM, Reumaux D, Roesler J, Schulze I, Wahn V, Papayannopoulos V, Zychlinsky A: Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity. Blood. 2010, 117: 953-959.PubMedView ArticleGoogle Scholar
- Jirousek MR, Gillig JR, Gonzalez CM, Heath WF, McDonald JH, Neel DA, Rito CJ, Singh U, Stramm LE, Melikian-Badalian A: (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e, k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta. J Med Chem. 1996, 39: 2664-2671. 10.1021/jm950588y.PubMedView ArticleGoogle Scholar
- Dekker LV, Leitges M, Altschuler G, Mistry N, McDermott A, Roes J, Segal AW: Protein kinase C-beta contributes to NADPH oxidase activation in neutrophils. Biochem J. 2000, 347 (Pt 1): 285-289.PubMedPubMed CentralView ArticleGoogle Scholar
- Doussiere J, Vignais PV: Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation. Eur J Biochem. 1992, 208: 61-71. 10.1111/j.1432-1033.1992.tb17159.x.PubMedView ArticleGoogle Scholar
- Segal AW: How neutrophils kill microbes. Annu Rev Immunol. 2005, 23: 197-223. 10.1146/annurev.immunol.23.021704.115653.PubMedPubMed CentralView ArticleGoogle Scholar
- Babior BM, Lambeth JD, Nauseef W: The neutrophil NADPH oxidase. Arch Biochem Biophys. 2002, 397: 342-344. 10.1006/abbi.2001.2642.PubMedView ArticleGoogle Scholar
- Yost CC, Cody MJ, Harris ES, Thornton NL, McInturff AM, Martinez ML, Chandler NB, Rodesch CK, Albertine KH, Petti CA: Impaired neutrophil extracellular trap (NET) formation: a novel innate immune deficiency of human neonates. Blood. 2009, 113: 6419-6427. 10.1182/blood-2008-07-171629.PubMedPubMed CentralView ArticleGoogle Scholar
- Urban CF, Reichard U, Brinkmann V, Zychlinsky A: Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol. 2006, 8: 668-676. 10.1111/j.1462-5822.2005.00659.x.PubMedView ArticleGoogle Scholar
- Bianchi M, Hakkim A, Brinkmann V, Siler U, Seger RA, Zychlinsky A, Reichenbach J: Restoration of NET formation by gene therapy in CGD controls aspergillosis. Blood. 2009, 114: 2619-2622. 10.1182/blood-2009-05-221606.PubMedPubMed CentralView ArticleGoogle Scholar
- Ermert D, Urban CF, Laube B, Goosmann C, Zychlinsky A, Brinkmann V: Mouse neutrophil extracellular traps in microbial infections. J Innate Immun. 2009, 1: 181-193. 10.1159/000205281.PubMedView ArticleGoogle Scholar
- Papayannopoulos V, Zychlinsky A: NETs: a new strategy for using old weapons. Trends Immunol. 2009, 30: 513-521. 10.1016/j.it.2009.07.011.PubMedView ArticleGoogle Scholar
- Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, Nacken W, Brinkmann V, Jungblut PR, Zychlinsky A: Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog. 2009, 5: e1000639-10.1371/journal.ppat.1000639.PubMedPubMed CentralView ArticleGoogle Scholar
- Lande R, Ganguly D, Facchinetti V, Frasca L, Conrad C, Gregorio J, Meller S, Chamilos G, Sebasigari R, Riccieri V: Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. Sci Transl Med. 2011, 3: 73ra19-10.1126/scitranslmed.3001180.PubMedPubMed CentralView ArticleGoogle Scholar
- Leffler J, Martin M, Gullstrand B, Tyden H, Lood C, Truedsson L, Bengtsson AA, Blom AM: Neutrophil extracellular traps that are not degraded in systemic lupus erythematosus activate complement exacerbating the disease. J Immunol. 2012, 188 (7): 3522-3531. 10.4049/jimmunol.1102404.PubMedView ArticleGoogle Scholar
- Garcia-Romo GS, Caielli S, Vega B, Connolly J, Allantaz F, Xu Z, Punaro M, Baisch J, Guiducci C, Coffman RL: Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus. Sci Transl Med. 2011, 3: 73ra20-10.1126/scitranslmed.3001201.PubMedPubMed CentralView ArticleGoogle Scholar
- Manzenreiter R, Kienberger F, Marcos V, Schilcher K, Krautgartner WD, Obermayer A, Huml M, Stoiber W, Hector A, Griese M: Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy. J Cyst Fibros. 2012, 11 (2): 84-92. 10.1016/j.jcf.2011.09.008.PubMedView ArticleGoogle Scholar
- Marcos V, Zhou Z, Yildirim AO, Bohla A, Hector A, Vitkov L, Wiedenbauer EM, Krautgartner WD, Stoiber W, Belohradsky BH: CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation. Nat Med. 2010, 16: 1018-1023. 10.1038/nm.2209.PubMedView ArticleGoogle Scholar
- Lin AM, Rubin CJ, Khandpur R, Wang JY, Riblett M, Yalavarthi S, Villanueva EC, Shah P, Kaplan MJ, Bruce AT: Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis. J Immunol. 2011, 187: 490-500. 10.4049/jimmunol.1100123.PubMedPubMed CentralView ArticleGoogle Scholar
- Kong L, Andrassy M, Chang JS, Huang C, Asai T, Szabolcs MJ, Homma S, Liu R, Zou YS, Leitges M: PKCbeta modulates ischemia-reperfusion injury in the heart. Am J Physiol Heart Circ Physiol. 2008, 294: H1862-H1870. 10.1152/ajpheart.01346.2007.PubMedView ArticleGoogle Scholar
- Leitges M, Schmedt C, Guinamard R, Davoust J, Schaal S, Stabel S, Tarakhovsky A: Immunodeficiency in protein kinase cbeta-deficient mice. Science. 1996, 273: 788-791. 10.1126/science.273.5276.788.PubMedView ArticleGoogle Scholar
- Skvara H, Dawid M, Kleyn E, Wolff B, Meingassner JG, Knight H, Dumortier T, Kopp T, Fallahi N, Stary G: The PKC inhibitor AEB071 may be a therapeutic option for psoriasis. J Clin Invest. 2008, 118: 3151-3159. 10.1172/JCI35636.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.