Introduction
Neutrophil granulocytes, also known as polymorphonuclear leukocytes (PMN), are the largest population among the cells of the innate immune system and an essential component of the first line of defense against invading pathogens. Once PMN arrive at the site of infection, they employ different strategies to kill pathogens.
1 Primarily, PMN engulf microorganisms and ingest them by phagocytosis. Upon internalization of pathogens into phagocytic vesicles, the latter are fused with lysosomes containing proteins with antimicrobial activity. These proteins include NADPH oxidase, an enzyme capable of producing high amounts of reactive oxygen species (ROS).
PMN extend their antimicrobial activity even beyond their life by a newly described strategy in which they cast out their DNA, chromatin and granule proteins into the extracellular space, forming a matrix known as neutrophil extracellular traps (NET).
2 These NETs capture and kill bacteria, contributing to clearance of the infection. This specialized form of cell death, so-called NETosis, is NAPDH oxidase-dependent,
3 and differs both biochemically and morphologically from necrosis and apoptosis.
4 Several different triggers for NETosis have been identified, including bacteria, LPS and phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC).
3 The latter is a potent activator of NADPH oxidase. This enzyme produces superoxide anions, which can be converted into H
2O
2. Notably, exogenous H
2O
2 can promote NET production by PMN isolated from chronic granulomatous disease patients, whose cells lack functional NAPDH oxidase.
4
PMN have a short lifespan, are terminally differentiated and there is considerable variation in their ability to produce NETs between different donors.
1,4 Moreover, the isolation of primary PMN is time consuming and might cause pre-activation of the cells, complicating the analysis of NETosis.
4 Accordingly, the identification of a human cell line with neutrophil characteristics as a model for NET formation would provide a valuable alternative to primary PMN in the investigation of NETosis.
The cell line NB4, established from a patient with acute promyelocytic leukemia,
5 shows bilineage potential.
6 It undergoes differentiation into morphologically mature PMN in response to all-trans retinoic acid (ATRA) and DMSO, or toward monocytes after exposure to 1α,25-dihydroxyvitamine D3 (1,25D3) and PMA.
6,7 N’Diaye and colleagues have reported that ATRA-differentiated NB4 cells express functional NAPDH oxidase and exhibit respiratory burst activity in response to PMA,
8 suggesting that they might have the potential for NET formation.
Zinc was identified as an essential trace element in humans in the early 1960s.
9 In addition to several other biological functions it is indispensible for the immune system; zinc deficiency is associated with an increased risk of infections.
10 Among the cells affected by zinc deficiency are PMN. Here, chemotaxis and the production of ROS were reported to be affected.
11 Notably, the impact of zinc on NET formation has not been investigated so far.
The essentiality of zinc is based partially on its role as a structural component of a great number of proteins, including more than 300 enzymes and a multitude of transcription factors.
12 Consequently, zinc homeostasis is tightly regulated by zinc-binding molecules, such as metallothionein, and two dozen zinc transporters, which mediate Zn
2+ transfer through the plasma membrane and between intracellular compartments.
13 Additionally, alternations in the intracellular concentration of free Zn
2+ occur, so-called zinc signals. These are induced by extracellular stimuli and participate in several signaling pathways in immune cells.
14 One of these stimuli is PMA, which causes zinc signals in monocytes, suggesting that some signaling pathways downstream of PKC could involve Zn
2+.
15
The aim of this study was to investigate a potential role of free intracellular Zn2+ in PKC-mediated NET formation. To this end, NB4 cells differentiated along the neutrophilic lineage were established as a cell culture model for NET formation in response to stimulation with PMA. In these cells, PMA-induced Zn2+ signals were observed and were found to be essential, but not sufficient, for NETosis.
Materials and methods
Materials
RPMI-1640 cell culture medium, pencillin, streptomycin, L-glutamine, sodium pyruvate and PBS were purchased from Lonza (Verviers, Belgium). Low endotoxin FCS was obtained from PAA (Coelbe, Germany) and was heat-inactivated for 30 min at 56℃ prior to use. ATRA, N,N,N′,N′-tetrakis-2(pyridyl-methyl)ethylenediamine (TPEN), PMA, Hoechst 33258 and sodium pyrithione were from Sigma-Aldrich (Taufkirchen, Germany). Calcitriol (1α,25-dihydroxyvitamin D3; 1,25D3) was obtained from Tocris bioscience (Ellisville, MO, USA) and dissolved in ethanol (absolute); further dilutions were made in RPMI 1640. ZnSO4 × 7 H2O purchased from Merck (Darmstadt, Germany). All other chemicals were from standard sources and of analytical quality.
Isolation of primary human cells
Primary human leukocytes were isolated from heparinized whole blood of healthy, consenting donors (ethical approval for the use of blood from human volunteers had been obtained from the institutional ethics review board of RWTH Aachen University Hospital under protocol number EK 023/05). One part of a 6% hydroxyethyl starch solution was added to two parts of blood and sedimentation for 45–60 min at room temperature (20–22℃) was followed by two washes with PBS and hypotonic lysis of remaining erythrocytes.
Cell culture and differentiation
The human promyelocytic cell line NB4 was cultured at 37℃ in a humidified 5% CO2 atmosphere. Cells were grown in RPMI-1640 containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 1 mM sodium pyruvate. For neutrophil differentiation, NB4 (2 × 105/ml) were incubated in culture medium supplemented with ATRA (0.3 µM) in the presence of 0.3% (v/v) DMSO for 4 d. For monocyte differentiation, NB4 cells were seeded at a density of 4 × 105/ml and treated with 100 nM 1,25D3 in combination with 0.6 ng/ml PMA for 2 d. Cellular viability was monitored by trypan blue exclusion.
Measurement of cell surface markers
NB4 cells were taken up in PBS and incubated with anti-CD66b FITC-conjugated Ab (clone G10F5) and anti-CD14 PE-conjugated Ab (clone MøP9). Mouse IgMκ-FITC and mouse IgG2bκ-PE were used as isotype-matched controls (all Abs were from BD PharMingen, Heidelberg, Germany). Abs and cells were incubated for 30 min at room temperature in the dark, and washed once with PBS. Fluorescence was measured with a FACScan (Becton Dickinson, Heidelberg, Germany).
Fluorescence microscopy
Cells were activated with PMA (50 ng/ml) at 37℃ in culture medium. After 3 h Hoechst 33258 was added to a final concentration of 10 µg/ml, followed by further incubation for 1 h. Subsequently, cells were transferred onto glass slides by cytospin at 300 g for 5 min. Fluorescence was monitored with a Zeiss Axiskop and images were taken at 100-fold magnification using a Nikon Coolpix 4500 digital camera.
Quantification of NET formation
The release of NET was measured by the enhanced fluorescence emission of PI after its interaction with extracellular DNA.
16 To this end, cells were taken up either in PBS or measurement buffer (25 mM HEPES, pH 7.35, 120 mM NaCl, 5.4 mM KCl, 5 mM Glc, 1.3 mM CaCl
2, 1 mM MgCl
2, 1 mM NaH
2PO
4) and seeded into 96-well plates. Subsequently, cells were activated with PMA (50 ng/ml) or H
2O
2 (1 mM) at 37℃ for 4 h, followed by incubation with propidium iodide (PI, 10 µg/ml) for 5 min in the dark. The resulting fluorescence was recorded on a Tecan Ultra 384 fluorescence well plate reader using an excitation wavelength of 360 nm and emission wavelength of 612 nm
Free Zn2+ measurement
Free intracellular Zn
2+ was measured as described previously.
15 Briefly, cells were loaded with 1 µM FluoZin-3 acetoxymethyl ester for 30 min at 37℃ and their fluorescence was measured by flow cytometry. Free zinc concentrations were calculated using a dissociation constant for the Zn
2+/FluoZin-3 complex of 8.9 nM,
17 determining the minimal and maximal fluorescence by addition of 50 µM TPEN or a combination of Zn
2+ (100 µM) and the ionophore pyrithione (50 µM).
Determination of ROS production
Cells were taken up in PBS and incubated with 1 µg/ml DHR 123 (Invitrogen, Karlsruhe, Germany) for 30 min at 37℃. Afterwards, cells were washed once with PBS and the oxidation of DHR 123 was analyzed by flow cytometry.
Immunoblotting
After incubation as indicated in the figure legend, cells were collected by centrifugation and lysed in sample buffer [6.25 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% glycerol, 0.1% (v/v) β-mercaptoethanol, 0.01% (w/v) bromophenol blue, and 1 mM sodium orthovanadate], sonicated for 10 s and boiled for 5 min at 95℃. An equivalent of 1.5 × 105 cells per lane was separated on 10% polyacrylamide gels at 160 V and blotted onto nitrocellulose membrane (BioRad, Munich, Germany). Uniform loading of gels was confirmed by staining with Ponceau S (Sigma-Aldrich). After destaining, membranes were blocked with 5% fat-free dry milk in TBS-T [20 mM Tris-HCl, pH 7.6, 136 mM NaCl, 0.1% (v/v) Tween 20] for at least 1 h, followed by incubation at 4℃ with a primary Ab against phospho-(Ser) PKC substrate or β-actin, gently shaking overnight (18–20 h; both Abs and biotinylated protein ladder were obtained from Cell Signaling Technology, Frankfurt, Germany). Subsequently, membranes were washed three times with TBS-T and incubated at room temperature for at least 1 h with HRP-linked anti-rabbit IgG secondary Ab and HRP-coupled anti-biotin Ab, followed by detection with LumiGlo reagent (Cell Signaling Technology) on a LAS-3000 (Fujifilm Lifescience, Düsseldorf, Germany).
Statistical analysis
Statistical significance of experimental results was calculated by one-way ANOVA followed by Tukey’s post hoc test using GraphPad prism software. All experiments were performed independently at least three times.
Discussion
Zinc deficiency negatively affects human health, particularly through its impact on the immune system. Without a sufficient supply with zinc, defense against infectious agents is impossible; infections as a result of zinc deficiency are a major cause of the loss of healthy life years.
18 This is based on two biological roles of zinc: (i) its role as a prosthetic group in proteins, and (ii) a function of free Zn
2+ in signal transduction. Several examples for the latter have been reported, including the regulation of the activity of monocytes, dendritic cells, mast cells, and T cells.
14 PMN are also functionally impaired in zinc deficient individuals, but a function of Zn
2+ in their signal transduction still remains to be shown.
One particular function of PMN—NETosis—which was discovered a few years ago, is still poorly understood with regard to the intracellular mechanisms by which it is regulated. The most frequently used pathway of induction is triggered by PMA, a PKC activator. PKC, in turn, activates the NADPH oxidase complex. It produces superoxide anions, the starting product of the ROS that constitute the oxidative burst, a mechanism by which PMN kill phagocytosed pathogens. In addition, it was shown that these ROS are also required to trigger the release of NETs, consisting of DNA, chromatin and antibacterial granule proteins, forming a matrix in which extracellular pathogens are captured and killed.
3
NB4 cells are a pro-myeloid cell line that have retained the ability to develop into monocytes or PMN.
6 For neutrophil differentiation, this includes expression of functional NAPDH oxidase,
8 the starting point of the signals that trigger NETs. The data in
Figure 1 demonstrate that by differentiating into PMN, NB4
PMN cells have gained the ability to undergo PMA-dependent NETosis, which is blocked by inhibition of NADPH oxidase. In contrast, no NETosis is observed in undifferentiated NB4 and NB4
MONO cells, demonstrating that NB4
PMN cells are a suitable and specific cell culture model for investigation of NETosis.
An increase in the intracellular concentration of free Zn
2+ has been observed in T cells stimulated by phorbol ester.
19 PMA has also been reported to cause zinc signals in monocytes, a cell type which develops from the same precursor as neutrophils.
15 A comparable zinc signal was also found in NB4
PMN cells and primary human PMN, indicating that Zn
2+ could be involved in the signal transduction leading to NETosis.
Inhibition of zinc signals by the NADPH oxidase inhibitor DPI suggests that Zn
2+ is released as a consequence of ROS production. This is in contrast to reports in several other signaling contexts, in which zinc is acting further upstream, activating PKC. Activation of this kinase was inhibited by the zinc chelator TPEN,
20,21 but the concentrations used for inhibition (up to 100 µM) were significantly higher than the 5 µM or less that were used in the present study. Moreover, addition of exogenous Zn
2+ to cell cultures induced PKC translocation to the cytoskeleton, which is part of its activation.
22 As shown in
Figure 5, production of ROS was unaffected by the same concentrations of TPEN that inhibited NETosis. Furthermore, PMA-mediated phosphorylation of several proteins containing a PKC target sequence, which was taken as a measure for PKC activity, was also unchanged by TPEN. This shows that the rise in free Zn
2+ in response to stimulation with PMA is a consequence of PKC-mediated NADPH oxidase activation, but is not required for activation of PKC. The source of the Zn
2+ remains unknown; it has been demonstrated that ROS can release Zn
2+ from Zn
2+-binding proteins such as metallothionein
23 or PKC itself.
24 This is clearly different from other signaling pathways, such as IL-2-signaling in T cells, during which lysosomal Zn
2+ is released.
25,26
Investigation of the time course in
Figure 7 showed that TPEN blocks NET formation even if added up to 30 min after PMA, which is the timepoint at which the Zn
2+ measurements in
Figures 3,
4 and
5 showed elevated levels of free intracellular Zn
2+. TPEN enters cells within seconds;
15 therefore, it should chelate the zinc signal immediately after its addition. This indicates that free Zn
2+ is required during NETosis between 30 and 60 min after PKC activation. These data support a sequence of events in which PKC triggers production ROS, which, subsequently, raise intracellular Zn
2+.
An effect of TPEN downstream of its conventional effect on PKC signaling calls for an investigation if it is really acting as a Zn
2+ chelator, or, potentially, as a low molecular mass inhibitor independent of its metal chelating ability. The latter effect has already been observed in TPEN-induced apoptosis of PC12 cells.
27 Application of a Zn
2+/TPEN complex has no impact on NETosis, showing that TPEN is ineffective when it can no longer bind cellular metal ions because it is already saturated. This was not observed for TPEN complexes of Ca
2+ or Mg
2+, demonstrating that chelation of these two ions is not the molecular basis for the effect of TPEN. However, addition of Cu
2+ also prevented TPEN from inhibiting NETosis, raising the question of whether TPEN acts via chelation of Zn
2+ or Cu
2+. The affinity of TPEN for Cu
2+ is about five orders of magnitude stronger than the one for Zn
2+ (
= 4 × 10
15 M
−1,
= 3 × 10
20 M
−1).
28 Consequently, unless Zn
2+ is present in vast excess, TPEN will no longer bind this ion once it chelates Cu
2+. In contrast, Cu
2+ could still displace Zn
2+ from TPEN. Therefore, if TPEN would inhibit NETosis by chelation of Cu
2+, Zn
2+/TPEN should still have inhibitory activity. This consideration is further supported by measurement of free Zn
2+. FluoZin-3, which is insensitive toward Cu
2+ (data not shown), exhibits no significant diminution of the free Zn
2+ in response to Cu
2+/TPEN, demonstrating that this form of TPEN is unable to bind Zn
2+. Hence, our data show that TPEN inhibits NETosis through its metal binding capability, namely by binding Zn
2+.
The high affinity of TPEN for Zn
2+ raises the possibility that, instead of chelating free Zn
2+, the chelator could inhibit NETosis by removal of Zn
2+ from a critical protein, which subsequently loses its function. However, after addition of 5 µM TPEN (
Figure 5C), there still was some free intracellular Zn
2+ left for detection by FluoZin-3, which has a nanomolar dissociation constant for this ion.
17 Thermodynamically, a chelator will prefer the pool of Zn
2+ that is bound with the lowest affinity. Hence, TPEN will not remove tightly bound Zn
2+ from proteins, unless all free (and FluoZin-3-bound) ions are already chelated. Notably, in the literature more than 100 µM of TPEN were required to remove protein-bound Zn
2+ from a zinc finger structure.
29 Additionally, as shown in
Figure 5, no effect of 5 µM TPEN on PKC activation was observed. As mentioned above, higher concentrations of TPEN do inhibit PKC.
20,21
Addition of H
2O
2 to NB4
PMN cells confirms that ROS are sufficient for triggering NETosis, as reported by Fuchs et al.
4 As for PMA-stimulated NETosis, a zinc signal is also involved if H
2O
2 is used for inducing NETs. Chelation of Zn
2+ abrogates NETosis, demonstrating that zinc signals are essential for the signal transduction leading to NET formation in response to H
2O
2, as well as PMA. However, when an intracellular zinc signal was generated by application of extracellular Zn
2+ in the presence of the ionophore pyrithione, no NETosis was observed. Hence, simply raising the intracellular Zn
2+ concentration is not sufficient to trigger NETs in the absence of ROS. The time course after treatment with Zn
2+ and pyrithione may be different from the physiological course of the zinc signal and therefore unsuitable to cause NETosis. Alternatively, it is very likely that Zn
2+ is only one second messenger acting in concert with other ROS-derived signals.
In conclusion, zinc signals are utilized by PMN. In the present example, PKC activates NADPH oxidase, which synthesizes ROS that release Zn
2+. This Zn
2+ is then one of several ROS-derived signals leading to NETosis. It remains to be identified which molecular targets interact with Zn
2+ in the downstream signal transduction. So far, only some parts of the mosaic of signaling pathways involved in NETosis have been identified. Many different signal transduction pathways in immune cells are known to involve Zn
2+,
14 and it will have to be determined which of them are involved in the formation of NETs and which one(s) are actually responsible for the observed zinc-dependence.