Aus der Klinik für Anästhesiologie und Intensivmedizin der Medizinischen Hochschule Hannover
Regulation der Adhäsion, Transmigration und Chemotaxis von neutrophilen Granulozyten der Maus in vitro durch die
Natriumkanal- α -Untereinheit Na
v1.3
Dissertation
zur Erlangung des Doktorgrades der Medizin in der Medizinischen Hochschule Hannover
vorgelegt von
Marit Poffers aus Nordhorn
Angenommen vom Senat der Medizinischen Hochschule Hannover am 02.06.2020
Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover
Präsident
Prof. Dr. med. Michael P. Manns
Betreuer der Arbeit
Prof. Dr. med. Andreas Leffler
Referenten
Prof. Dr. med. Florian Wegner Prof. Dr. med. Matthias Eder
Tag der mündlichen Prüfung 02.06.2020
Prüfungsausschussmitglieder
Vorsitz: Prof. Dr. med. Hermann Müller-Vahl
1. Prüfer: Prof. Dr. med. Marc Ziegenbein 2. Prüfer: Prof. Dr. med. Frank Schuppert
Inhaltsverzeichnis
I. Originalarbeit 2
I.1. Abstract 3
I.2. Introduction 4
I.3. Materials & Methods 6
I.4. Results 14
I.5. Discussion 25
I.6. Supplementary Figures 29
I.7. References 31
II. Zusammenfassung 36
II.1. Einleitung 36
II.2. Diskussion 41
II.3. Abkürzungen 47
II.4. Literaturverzeichnis 47
III. Angaben zur Publikation 52
IV. Lebenslauf 54
V. Danksagung 57
VI. Erklärung nach § 2 Abs. 2 Nrn. 6 und 7 PromO 58
I. Originalarbeit
publiziert in: Anesthesiology, 2018; 128:1151-66
The sodium channel Nav1.3 is expressed by polymorphnuclear neutrophils during mouse heart and kidney ischemia in vivo and regulates adhesion, transmigration and chemotaxis of human and mouse neutrophils in vitro
Marit Poffers, cand. med.1, Nathalie Bühne, cand. med.1, Christine Herzog, Ph.D.1, Anja Thorenz, Ph.D.2, Rongjun Chen, MSc2, Faikah Gueler, M.D.2, Axel Hage, M.D.1, Andreas Leffler, M.D.1, Frank Echtermeyer, Ph.D.1#
Department of Anesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany
I.1. Abstract
Background: Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed non-canonical roles in non-excitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils.
Method: Expression of voltage-gated sodium channels was analyzed by polymerase- chain-reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with whole-cell patch clamp.
Results: Mouse and human neutrophils express multiple voltage-gated sodium channels.
Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7 %, n = 14) and kidney (19 ± 2 %, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9 %, unselective Nav-inhibitor), ICA121431 (53 ± 10 %) and Pterinotoxin-2 (55 ± 9 %) (preferential inhibitors of Nav1.3, n = 10).
Tetrodotoxin (56 ± 19 %), ICA121431 (62 ± 22 %) and Pterinotoxin-2 (59 ± 22 %) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3x 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9 % (n = 10), and transmigration to 54 ± 8 % (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2.
Conclusion: Nav1.3 is expressed in neutrophils in vivo, regulates attachment, transmigration and chemotaxis in vitro and may serve as a relevant target for anti- inflammatory effects of lidocaine.
I.2. Introduction
All nine known pore forming a-subunits of voltage-gated sodium channels (VGSC) are regarded to dictate action potential generation in excitable cells [1]. Accordingly, inhibi- tion or modulation of excitability by local anesthetics seems to be primarily due to an inhibition of VGSCs [2]. While the expression pattern of most a-subunits suggests that they have rather specialized tissue-specific functions [1], recent studies show that so called “neuronal” or “cardiac” α-subunits are expressed in a variety of tissues and that some α-subunits even seem to be expressed in non-excitable cells including tumor cells, glia and immune cells [3]. While non-excitable cells obviously do not generate action potentials, VGSC α-subunits were reported to perform noncanonical roles and thus regu- late diverse cellular functions including cell motility, migration, invasiveness and phagocytosis to name some [3]. In macrophages, the cardiac α-subunit Nav1.5 regulates endosomal acidification, phagocytosis and degradation of myelin in multiple sclerosis (MS) lesions and possibly acts as pathogen sensor [4-6]. Moreover, Nav1.5 regulates migration and proliferation of astrocytes after mechanical injury through mechanisms involving a [Ca2+] transient [7]. A more recent study suggested that inhibition of several VGSC α-subunits expressed in macrophages and/or monocytes by phenytoin improves cardiac function following cardiac ischemia and reperfusion in adult rats [8]. The skeletal muscle α-subunit Nav1.4 was reported to play a crucial role for positive selection of CD4-positive T-cells [9], and the sensory neuronal α-subunit Nav1.7 regulates migration and cytokine responses of dendritic cells [10] and migration of endothelial cells [11].
These noncanonical roles of VGSCs in different immune cells indicate that VGSC-inhibitors should have anti-inflammatory properties. Indeed, lidocaine and other local anesthetics were shown to induce anti-inflammatory and anti-apoptotic effects in both rodent and human studies [12-15]. However, this interesting property of primarily lidocaine is usually attributed to a modulation of PMN-function including a reduction of activation, priming and recruitment [12, 14, 16]. While several molecular mechanisms have been suggested to account for these lidocaine-induced effects, they are commonly stated to be VGSC-independent. This statement is probably given mainly because effects on PMNs occur at low micromolar (< 10 µM) concentrations of lidocaine which are not sufficient to robustly inhibit sodium currents generated by VGSCs. As inhibition of VGSCs by local anesthetics obeys a strong state-dependency however, very low
concentrations of lidocaine can inhibit sodium currents at depolarized membrane poten- tials [2].
Being alerted by the increasing number of studies demonstrating diverse noncanonical roles of VGSC α-subunits in immune cells other than PMNs, we hypothesized that VGSCs are relevant for PMN-function and for lidocaine-induced effects on PMNs.
Employing a variety of in vitro and in vivo assays on mouse and human PMNs, we found that the neuronal α-subunit Nav1.3 is expressed in a fraction of PMNs both in vitro and in vivo. Furthermore, pharmacological inhibition of Nav1.3 strongly abbreviated attachment, transmigration and chemotaxis of PMNs. We therefore suggest that Nav1.3 might be a relevant target for anti-inflammatory effects of VGSC-blocking agents like lidocaine.
I.3. Materials & Methods
Mouse model of myocardial ischemia with reperfusion injury
This study was approved by the Institutional Review Board and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US Na- tional Institutes of Health. For myocardial ischemia with reperfusion (MI/R) injury, the left coronary artery of 8 - 10 weeks old C57BL/6 mice was ligated for 30 minutes fol- lowed by 24 h and 72 h of reperfusion. An average infarct size of the area at risk of 47.5 % was confirmed by TTC/Coomassie staining as shown earlier [17]. For histological analysis, hearts were harvested and cryo-embedded at 24 h or 72 h after surgery. For kidney ischemia 12 - 14 weeks old male C57BL/6 mice were anesthesized with isofluran (3 % for induction and 1.5 % for maintenance) and butorphanol 1 mg/kg was given s.c. for analgetic treatment. The left renal pedicle was clamped for 45 minutes to induce unilateral ischemia reperfusion injury. Mice were sacrificed 24 hours after IRI by total body perfusion with ice cold PBS via the left ventricle in deep general anesthesia resulting in circulatory arrest. One part of the tissue was shock frozen for immuno- histochemistry the rest was rinsed in PBS and used for leukocyte isolation.
Isolation of PMNs
Mouse PMNs were freshly isolated from bone marrow using a modified protocol described by Zhang et al [18]. C57BL/6 mice were euthanized by cervical dislocation.
Femur and tibia were separated, soft tissues were removed with scalpel and bone marrow cells were flushed out with approximately 5 ml of HBSS supplemented with 0,1 % BSA per bone using a 20 G needle. Cell suspension was dispersed with a 18 G needle, filtered through cell strainers (40 µm, Becton Dickinson, Bedford, MA), centrifuged for 5 minutes at 2200 rpm and erythrocytes were removed by hypotonic lysis. After centrifugation cells were resuspended in HBSS or medium, depending on the type of assay, and cell viability determined by trypan blue exclusion.
Human PMNs were isolated from peripheral blood obtained from healthy volunteers in accordance with the Hannover Medical School Institutional Review Board. Briefly, EDTA-anticoagulated blood was layered on top of a 1.077 g/ml Biocoll (Biochrom,
Berlin, Germany) density gradient and after centrifugation at 800 g for 20 minutes the PMN containing band was collected. Erythrocytes were removed by lysis with cold ddH2O. PMNs were resuspended in HBSS, cell viability determined by trypan blue exclusion and a purity of > 95 % verified by Maygrünwald/Giemsa staining of cytospins.
RT-PCR
Expression of voltage gated sodium channels (VGSCs) was analyzed by RT-PCR as described earlier [17]. Briefly, total RNA from PMNs attached to cell culture plastic for 24 h, 4 h or 15 min with or without TNFα stimulation as indicated was extracted with Tritidy reagent (Applichem, Darmstadt, Germany) and 500 ng RNA was used for cDNA synthesis (Reverse Transcription Core Kit, Eurogentec, Cologne, Germany). Primer for the different VGSC isoforms are listed in table S1 (Supplementary material). PCR was performed on a real time PCR cycler (Rotorgene 3000, Corbett Life Science, Hilden, Germany). Signals were generated by SYBR green incorporation (SensiFAST™ SYBR No-ROX, Bioline, Luckenwalde, Germany) into the amplified DNA (40 cycles), and visualized on a 2 % agarose gel using UV-light in a Bio-Vision gel documentation system (Vilbert Lourmat, Eberhardzell, Germany).
Immunohistochemistry
Immunohistochemistry of cryo-embedded myocardial and ischemic kidney tissues was performed as described before [19]. Briefly, 5 µm cryo sections of myocardial tissue 24 h and 72 h after MI/R and 24 h after 45 minutes of kidney clipping were stained with primary antibodies against F4/80 (1:100, Bio-Rad AbD Serotec, Puchheim, Germany) for detection of macrophages or with GR-1 (1:100, Bio-Rad AbD Serotec, Puchheim, Germany) or Ly6B (clone 1A8, 1:100, BioLegend, Fell, Germany) for identification of PMNs and with Nav1.3 (1:500, ASC004 and ASC023 Alomone labs, Jerusalem, Israel), all diluted in PBS supplemented with 0,1 % bovine serum albumin and 5 % normal donkey serum at 4 °C overnight. Secondary antibodies coupled to Cy3 or Cy5 (Jackson ImmunoResearch, Suffolk, UK) were diluted 1:100 in PBS and incubated for two hours
mounted with Fluorescent Mounting Medium (Biozol, Eching, Germany). After allowing mounted slides to dry completely, fluorescence was analyzed on an inverted fluorescence microscope (IX81, Olympus, Hamburg, Germany) using Q-Capture Pro7 software (QImaging, Surrey, Canada).
Western Blot
Specificity of Nav1.3 antibody was assessed by western blot analysis as described earlier [17]. Briefly, protein lysates were prepared in RIPA buffer including protease inhibitors (complete mini, no. 11836170001 Roche Diagnostics, Mannheim, Germany) from protein lysates of HEK 293 cells transfected with different sodium channels.
Protein amount was assessed by Pierce BCA protein assay kit (Thermo Scientific, Darmstadt, Germany). Proteins were separated by electrophoresis, transferred to PVDF membranes and identified by western blot using primary antibody Nav1.3 (Alomone labs, Jerusalem, Israel) diluted 1:100. Horseradish-peroxidase-coupled rabbit IgGs were used as secondary antibodies. Membrane was re-probed with anti-β-actin (no. 4967, Cell Signaling, Frankfurt, Germany) to correct for protein loading.
PMN Adhesion
Adhesion assays were performed as described [20]. Briefly, immortalized murine endothelioma cells or human umbilical vein endothelial cells (Lonza, Cologne, Germany), were grown to confluency in collagen-coated 6-well dishes in DMEM supplemented with 10 % FCS (f.End5) or EGMTM-2 Bullet-kitTM growth medium (Lonza, Cologne, Germany) with 5 % FCS (HUVEC), starved for 8 h in DMEM or EGMTM-2 Bullet-kitTM growth medium without FCS and stimulated with 10 ng/ml mouse or human TNFα (R&D Systems, Wiesbaden, Germany) for 12 h at 37 °C. Iso- lated mouse bone marrow PMNs or human whole blood PMNs were labeled with Cell Tracker Green (Molecular Probes, Eugene, USA) and 5x105 cells in 1 ml Binding Buffer (HBSS supplemented with 1 % BSA, 2 mM Ca2+ and 2 mM Mg2+) were allowed to adhere to activated endothelial cells in 6 well cell culture plates for one hour on a tilting table in the presence of 100 nM tetrodotoxin (Biotrend, Cologne, Germany), 1 µM
ICA121431 and 0,5 µM Pterinotoxin-2 (Alomone labs, Jerusalem, Israel) to block Nav1.3 channel activity as indicated. Afterwards, non-adherent cells were removed by extensive washing with HBSS. Each condition was performed in triplicates and eight randomly selected high power fields per well were documented with 200 x magnification on an inverted fluorescence microscope (IX81, Olympus, Hamburg, Germany) using Q- Capture Pro7 software (QImaging, Surrey, Canada). The number of adherent cells was determined using ImageJ version 1.50g (NIH, USA).
Flow Cytometry to estimate Nav1.3 expressing neutrophils
About 70 % of the renal tissue was used for leukocyte isolation as described previously [21]. Tissues were homogenized with a gentleMACS dissociator (Miltenyi Biotec, San Diego USA) according to manufacturer’s instructions. Digestion with Collagenase II (500 U/ml, Worthington, Lakewood, USA) was performed before and after homogenization for 20 minutes at 37 °C. A 70 µM cell strainer was used to obtain single cell suspensions. Incubation in red blood cell lysis buffer (420301, Biolegend, San Diego, USA) for one minute was done for erythrocyte lysis. For flow cytometry to analyze the proportion of Nav1.3 expressing neutrophils cells were gated by foreward scatter/ sideward scatter and by expression of CD45 (clone 30-F11, Biolegend, San Diego, CA) and fixable viability dye eFluor 506 (65-0866, ebioscience, Santa Clara, CA). Ly6G-FITC (clone 1A8, Biolegend, San Diego, CA) in combination with Nav1.3 (ASC023, Alomone labs, Jerusalem, Israel) was applied for detection of Nav1.3 expressing PMNs FACS CantoII (BD Biosciences, Heidelberg, Germany) was used for flow cytometry and data analysis was done using Kaluza software 1.3 (Beckmann Coulter, Krefeld, Germany).
Transmigration assay
Migration of PMNs across a dense layer of endothelial cells or the extracellular matrix protein collagen type I was performed in transwell chambers. 5 µm transwell filters (Costar, Omnilab, Hannover, Germany) were coated either with 50 mg/ml rat tail
and f.End5 or HUVEC cells that were grown to confluency in DMEM supplemented with 10 % FCS (f.End5) or EGMTM-2 Bullet-kitTM growth medium (Lonza, Cologne, Germany) with 5 % FCS (HUVEC), starved for 12 h in DMEM or EGMTM-2 Bullet- kitTM growth medium without FCS and stimulated with 10 ng/ml mouse or human TNFα (R&D Systems, Wiesbaden, Germany) for 4 h at 37 °C. Isolated mouse bone marrow PMNs or human whole blood PMNs were diluted to 5 x 105 in 100 µl DMEM and allowed to transmigrate through transwell filters coated with collagen type I, collagen type I and fEnd.5 cells or collagen type I and HUVECs towards 2 mg/ml N-Formyl-Met- Leu-Phe (Sigma, München, Germany) for two hours at 37 °C. Wells were either left untreated for control or treated with 100 nM TTX, 1 µM ICA121431, 0,5 µM Pterinotoxin-2, 10 µM, 100 µM or 1000 µM lidocaine (Sigma, München, Germany) or 100 µM lidocaine together with 100 nM TTX. Afterwards, cells migrated to the lower chamber were visualized by staining nuclei with DAPI and counted using a fluorescence microscope. Each condition was performed in duplicates or triplicates and 9 or 12 hpf/well were counted at 100 fold (mouse PMNs) or 40 fold (human PMNs) magnification.
Directed migration of PMNs
For analyzing Nav1.3-dependent differences in directionality of cell migration and cell velocity protocols of Peperrell et al. and Polesskaya et al. were used in a modified manner [22, 23]. Freshly isolated mouse bone marrow neutrophils were diluted to 3 x 106/ml in DMEM containing 10 % FCS and 6 µl were seeded to rat tail collagen type I-coated viewing channels of ibidi µ-Slides (Ibidi, München, Germany) with or without addition of 100 nM TTX, 1 µM ICA121431 and 0,5 µM Pterinotoxin-2, respectively. After allowing cells to adhere for 30 minutes at 37 °C in a humidified chamber followed by removal of nonadherent cells by washing, a fMLP gradient was established in the chambers by injecting 10 µM fMLP in DMEM supplemented with 10 % FCS to one of the two ports of the chemoattractant chamber. Neutrophils were allowed to migrate towards fMLP for 45 minutes at 37 °C prior to video analysis.
Observation of chemotaxis was performed recording videos over a time period of 15 minutes, saving a picture every 10 seconds with an Olympus IX81 microscope, Retiga
EXI fluorescence camera (QImaging, Surrey, Canada) and StreamPix Studio 6 software (NorPix Inc., Montreal, Canada). Cell tracking was performed using ImageJ plugin
„Manual Tracking“. Tracking data of a total of 60 cells (3 repeats with 20 cells each) for every experimental condition were analyzed with ImageJ plugin „Chemotaxis Tool“
(Ibidi, München, Germany) to derive trajectory plots and quantification data including accumulated migration distance, Euclidian distance, directionality of cell migration, forward migration indices, cell velocity and Rayleigh test results assessing distribution of migration data.
ROS-assay
Bone marrow PMNs were stained with 5 µM DCFH-DA (Invitrogen, Fisher Scientific, Germany) and this dye was allowed to diffuse into cells for 15 min at 37 °C in the dark.
Afterwards, baseline fluorescence was measured with ELISA using a Tecan Spectrafluor Plus. Cells were stimulated with 1 µM PMA and 2,5 x 105 cells/well were incubated in HBSS in a 96 well flat bottom plate (Greiner, Frickenhausen, Germany) with or without 100 nm TTX, 1 µM ICA 121431 and 0,5 µM Pterinotoxin-2. ROS-dependent increase in fluorescence was measured after 5, 10, 20, 30, 40, 50 and 60 minutes. Data are shown for 60 minutes only.
Whole-Cell Patch Clamp
Whole cell patch clamp experiments were carried out on bone marrow mouse and human peripheral blood PMNs as well as HEK 293 cells stably expressing rat Nav1.3 as described previously [24]. In addition Nav1.3 expressing mouse PMNs were isolated form ischemic kidneys 24 h after 45 minutes of IRI. Nav1.3 antibody (2 µg) was coupled to magnetic Protein G beads (100 µl, Milteny Biotech, Bergisch Gladbach, Germany) overnight at 4 °C and used to capture Nav1.3 expressing cells of cell lysates from one ischemic kidney each for one hour at room temperature. After washing with PBS containing 0.5 % BSA and 2 nM EDTA cells were cultivated in RPMI supplemented with 10 % heat-inactivated fetal bovine serum (Lonza, Cologne, Germany) and only
immediately used for patch clamp recording. HEK 293 cells were cultured in DMEM supplemented with 25 mM HEPES (Lonza, Cologne, Germany), 100 U/ml penicillin/streptomycin (Lonza, Cologne, Germany), 3 mM taurine (Sigma-Aldrich, München, Germany) and 10 % heat-inactivated fetal bovine serum (Lonza, Cologne, Germany). Cells were cultivated in cell culture flasks at 37 ºC and 5 % CO2. Membrane sodium currents were explored at room temperature using an EPC10 amplifier (HEKA Instruments Inc., NY, U.S.A.). Patch pipettes fabricates from glass capillaries (Science Products, Hofheim, Germany) on a DMZ-Universal Puller (Zeitz, Martinsried, Germany) and heat polished to give a resistance of 2.0 to 2.5 MΩ when filled with pipette solution.
The pipette solution contained (mmol/l): 140 CsF, 10 NaCl, 1 EGTA, 10 HEPES (pH 7.4). The external solution contained (mmol/l): 140 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 1 CaCl2, 10 HEPES and 15 glucose (pH 7.4). Voltage errors were minimized by compensation of the series resistance by 60 – 80 % and capacitance artefacts were cancelled using the amplifier circuitry. Currents were filtered at 5 kHz. Linear leak subtraction, based on resistance estimates from four hyperpolarizing pulses applied before the test pulse, was used for all voltage-clamp recordings other than use-dependent block. Data were acquired and stored with Patchmaster v20x60 software (HEKA Instruments Inc., NY, U.S.A.).
Statistics
Statistical analyses were conducted with Prism 7 (GraphPad Inc., San Diego, CA, USA).
Data that passed normality testing for distribution of data were tested with One-Way ANOVA and corrected with Bonferroni for multiplicity. Data that did not passed normality testing for distribution of data were tested with Kruskal-Wallis-test and were corrected with Dunns for multiplicity. For migration, Rayleigh test assessing inhomogenous distribution of data was performed with ImageJ Chemotaxis Tool (NIH).
A statistical power calculation to determine the number of experiments needed to achieve significant differences was not performed as all experiments are considered to be pilot experiments and numbers are based on previous experience. Two out of 12 sets of data for mouse adhesion assays had to be excluded since the difference in adherent PMNs between medium control and TNFα was not at least 1.5-fold and we had confirmed earlier that in this case endothelial cells had reached biological senescence, are no longer
activated by TNFα and could not be used for adhesion assays any more. For all adhesion, transmigration and directed migration assays data were blinded and experiment and data analysis were conducted by different persons. All values are presented as mean ± S.D.
and p < 0.05 was considered significant with the exception of data of PMN transmigration through collagen that did not pass normality testing and are therefore presented as median ± interquartile range.
I.4. Results
Functional expression of VGSCs in mouse and human PMNs
Over the past few years VGSCs have been shown to be expressed in several non- neuronal cells [3]. Based on these reports and the knowledge that unspecific blockers of VGSCs can modify PMN function [12, 16], we asked if VGSCs are expressed in mouse and human PMNs. In freshly isolated mouse bone marrow PMNs that were attached to cell culture plastic for 24 h we were able to show mRNA expression of Nav1.1 (very weak band), Nav1.3, Nav1.4, Nav1.5, Nav1.6 and Nav1.7 (weak band). In contrast, no expression of Nav1.2, Nav1.8 and Nav1.9 could be detected (Fig. 1A). In isolated PMNs from human blood however, all nine α-subunits but Nav1.1 seem to be expressed (Fig. 1B).
After having identified mRNA of multiple VGSCs α-subunits in both mouse and human PMNs, we investigated which of the VGSCs are detected on protein level on PMNs. Mouse PMNs attached to culture dish plastic for 24 h were stained with antibodies against different VGSCs. We only detected strong staining for Nav1.3 protein in some but not all GR-1 positive PMNs (Fig. 1C-E), whereas antibodies against Nav1.1, Nav1.5 and Nav1.7 showed no staining (not shown).
Next we investigated if cytokines like TNFα, IL-6 and IL-1β that activate PMNs during inflammation are able to Fig. 1. Expression of VGSCs in PMNs. (A, B)
Expression of VGSC mRNA in PMNs was analyzed by RT-PCR and amplified DNA fragments for α-subunits Nav1.1 - Nav1.9 (1.1 - 1.9) were visualized on a 2 % agarose gel of mouse bone marrow PMNs (A) and of human blood PMNs (B). (C-E) All mouse bone marrow PMNs attached to culture dish plastic for 24 h stained positive for the PMN marker GR-1 (C), whereas Nav1.3 is detected only on some attached PMNs (D) depicted by arrows in the merged picture in (E). (F) Nav1.3 mRNA expression is upregulated in PMNs after 4 h of attachment to cell culture plastic in the absence (medium 4h) or presence of TNFα (TNFα 4h) in comparison to control PMNs that were allowed to attach for 15 minutes (control 0h) only. Scale bar = 50 µm.
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Expression of VGSC α-subunits in mouse PMNs Expression of VGSC α-subunits in human PMNs
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upregulate Nav1.3 expression. Nav1.3 mRNA expression is enhanced 16-fold in PMNs attached to cell culture plastic for 4 hours compared to PMNs in suspension or PMNs attached for 15 minutes only (Fig. 1F). Stimulation with TNFα, interleukin-6 (not shown) or IL-1β (not shown) did not increase Nav1.3 expression of attached PMNs after 4 hours suggesting that adhesion and not inflammation is triggering Nav1.3 expression (Fig. 1F).
Nav1.3 seems to regulate adhesion- and migration-dependent functions of mouse and human PMNs
We next aimed to analyze the functional role of Nav1.3 during attachment of PMNs to activated endothelial cells, an early step of neutrophil recruitment. While Nav1.9, Nav1.8 and Nav1.5 are not blocked by the unselective VGSC blocker TTX, all other α-subunits are blocked by low nanomolar concentrations of TTX [25, 26]. In order to narrow down the effects to Nav1.3 using a pharmacological approach we combined the effects of TTX with that of ICA121431 (1.0 µM), a synthetic blocker of Nav1.1 and Nav1.3 [27] and Pterinotoxin-2 (0.5 µM), a synthetic analogon of a spider venom blocking Nav1.3 and Nav1.7. Although this combination of blockers cannot be used to specifically examine Nav1.3, Nav1.3 is the only VGSC α-subunit blocked by all three inhibitors and thus their effects on PMNs should deliver a good indication whether or not Nav1.3 is functionally expressed. Indeed, TTX significantly reduced mouse and human PMN adhesion rates to 56 ± 9 % and 54 ± 11 % respectively as compared to untreated cells. Furthermore, both 1.0 µM ICA121431 and 0.5 µM PTX-2 induced almost identical effects as TTX by reducing mouse PMN adhesion to 53 ± 10 % and 55 ± 9 % and human PMN adhesion to 50 ± 16 % and 50 ± 14 % respectively (Fig. 2A, B).
Furthermore, the majority of PMNs that were attached to activated endothelial cells were positively stained for Nav1.3 (Fig. 2C-F). The specificity of the antibody used to visualize Nav1.3 protein expression was verified by western blotting of protein lysates derived from HEK 293 cells transfected with cDNA for different VGSC. Only in Nav1.3 transfected HEK 293 cells a corresponding band with a molecular weight of 226 kDa could be observed, whereas Nav1.7 transfected and vector-transfected cells were negative (Supplement Fig. S1).
In a next step we asked if VGSCs and especially Nav1.3 are functionally expressed on PMNs that are recruited into inflamed tissue in vivo. First we stained mouse myocardial tissue cryosections obtained after myocardial ischemia for 30 minutes followed by reperfusion for 24 h or 72 h for Nav1.3 and the PMN marker GR-1 (24 h and low magnification of 72 h are shown in Supplement Fig. S2). Nav1.3 staining could be detected on individual cells within the infarcted area. High magnifications of 72 h MI/R sections revealed that Nav1.3 expression is co-localized with GR-1 in 25 ± 7 % of all GR-1 positive PMNs and only 3 ± 2 % of all cells were Nav1.3 positive and do not show staining for GR-1 (Fig. 3A, B).
Notably, immunochemistry with specific antibodies against Nav1.1, Nav1.5 and Nav1.7 in the same tissue sections only gave intense staining in cardiomyocytes outside the infarct area, but not in inflammatory cells (Supplement Fig. 3). Thus although we did not extensively rule out a role of other α-subunits in PMNs, it seems that the expression and function of Nav1.3 in PMNs does not apply for multiple α-subunits.
Fig. 2. Identification of Nav1.3 as the responsible VGSC α-subunit for reduced PMN adhesion. (A, B) Attachment of mouse (A) and human (B) PMNs to TNFα-activated endothelial cells (fEnd.5 for mouse and HUVEC for human PMNs) is significantly reduced after blocking VGSC α-subunits with TTX, ICA121431 and PTX-2 in comparison to medium (control) (**** p < 0.0001, n = 10).
(C-F) After adhesion of CTG-labeled PMNs to endothelial cells, PMNs were fixed with 4 % PFA and counterstained with DAPI to visualize nuclei of endothelial cells and PMNs (C). Incubation with antibodies against Nav1.3 showed that attached CTG-stained PMNs (D) show strong Nav1.3 expression (E) whereas Nav1.3 is not or very weak expressed by endothelial cells (F). Merged picture shows co-localization of CTG-stained PMNs and Nav1.3 expression (F). Scale bar = 50 µm.
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Fig. 3. Nav1.3 is expressed on neutrophils in ischemic mouse heart and kidney. (A) In ischemic mouse heart tissue 72 h (A, B, and C) after MI/R Nav1.3 (green staining) co-localizes with some (arrow heads) but not all GR-1 positive (red staining) neutrophils (arrows) indicated by yellow staining. (B) Fraction of cells within the infarcted area 72 h after MI/R that are positive for GR-1, Nav1.3 or double positive for GR-1 and Nav1.3 (n = 14). (C) No co-localization could be detected between Nav1.3 (green staining) and F4/80 positive macrophages (red staining) in 72 h MI/R sections and revealed only single positive cells for Nav1.3 (arrowheads) or F4/80 (arrows). (D) In ischemic kidney sections 24 h after 45 minutes IRI we detected Nav1.3 staining in some but not all GR-1 positive PMNs similar to the situation in MI/R. (E) Flow cytometry analysis of one single kidney revealed that 12.7 % of Ly6G high PMNs are also positive for Nav1.3, whereas 19.3 % of Ly6G low PMNs stained positive for Nav1.3. (F) Cell number of Ly6G and Ly6G/Nav1.3 double positive cells in ischemic kidneys (n = 6). (A, B, D) scale bar = 20 µm.
Since it has been reported that expression of VGSC on monocytes/macrophages modulates MI/R injury in rats [8, 28], we investigated if Nav1.3 staining occurs on macrophages in the infarcted area. However, neither 24 h nor 72 h after MI/R a co- localization between the pan-macrophage marker F4/80 and Na 1.3 could be observed,
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24h kidney I/R
E F
0 250000 500000 750000 1000000 1250000
cell number / 100mg kidney
Ly6G Ly6G
Nav1.3
105 105
104 104
103
103 102
102
Nav1.3-Cy3
Ly6G-FITC
1.0 42.2 54.3
2.5 101 101
(Fig. 3C and Supplement Fig. S2). Next we asked if Nav1.3 expressing PMNs are specific for MI/R or if Nav1.3 is present on PMNs in other inflammatory or ischemic organs as well. In a model of kidney ischemia 24 h after the left renal pedicle was clamped for 45 minutes to induce unilateral ischemia reperfusion injury, we could indeed confirm the MI/R results and demonstrated that Nav1.3 is expressed by a proportion of PMNs in ischemic kidney that was identified by GR-1 labeling in stained cryosections (Fig. 3D). Flow cytometry analysis of a single kidney in this model revealed that 12.7 % and 19.3 % of all PMNs are expressing Nav1.3 in Ly6G high and in Ly6G dim PMN populations, respectively (Fig. 3E). Overall flow cytometry analysis of multiple (n = 6) ischemic kidneys confirmed that 19.3 ± 2 % of all PMNs are expressing Nav1.3 and Ly6G (Fig. 3F). These data demonstrate a specific Nav1.3 staining which is associated with a fraction of PMNs in mouse heart and kidney tissue after ischemia pointing towards a common and important function of Nav1.3 during neutrophil recruitment.
Nav1.3 is important for transmigration of mouse and human PMNs in vitro
As we discovered PMNs expressing Nav1.3 and showed that we could reduce PMN adhesion with Nav1.3 specific blockers, we wanted to explore if Nav1.3 is influencing other PMN functions during tissue invasion. Therefore we addressed in the next step, if transmigration of PMNs through an endothelial cell layer and underlying basement membrane is altered by blocking Nav1.3 [29-31]. To mimic this in vitro, transmigration of mouse bone marrow PMNs and human peripheral blood PMNs through a confluent layer of f.End5 and HUVEC cells on collagen-coated Transwell-filters was recorded.
Medium supplemented with fMLP in the lower chamber was set as reference (100 ± 24 %; n = 6). Co-incubation with TTX (100 nM) significantly reduced rates of transmigrated PMNs to 63 ± 12 % (p = 0.0063, n = 6). The addition of ICA121431 and PTX-2 reduced transmigration rates to 66 ± 18 % and 65 ± 18 % respectively, again suggesting an important role for Nav1.3 in the transmigration process (Fig. 4A).
Fig. 4. Nav1.3 regulates PMN transmigration. (A) Transmigration of mouse bone marrow derived PMNs through an endothelial cell layer (fEnd.5) is significantly reduced when sodium channels were blocked with TTX or ICA121431 or PTX-2, where ICA121431 blocks Nav1.1 and Nav1.3 and PTX-2 blocks Nav1.3 and Nav1.7 (n = 6). (B) In order to avoid effects of sodium channel blockers related to endothelial cells, transmigration assay were performed on collagen-I coated transwell filter without endothelial cells. Addition of TTX, ICA121431 and PTX-2 significantly reduced the transmigration rate of PMNs through collagen as well (n = 8). Since data in the TTX treatment group in (B) did not pass normality test, data are tested by Kruskal-Wallis test followed by Dunn`s Multiple Comparison test and displayed as box blots showing median and 25 % and 27 % percentile and whiskers for 5 % and 95 % percentile. (C) In addition, Nav1.3 seems to regulate transmigration in humans as well since TTX, ICA121431 and PTX-2 significantly reduced transmigration of human blood derived PMNs through HUVECs (n = 9).
To eliminate possible effects of TTX, ICA121431 and PTX-2 by blocking VGSC expressed by endothelial cells, transmigration assays were repeated without f.End5 cells on Transwell-filters coated with collagen-I only. Transmigration on collagen-I matrices was reduced to 64 ± 30 % with TTX (n = 8), to 40 ± 24 % with ICA121431 (n = 9) and to 32 ± 15 % with PTX-2 (n = 9) compared to medium containing fMLP (control, set to 100 ± 9 %) (Fig. 4B, data are reported as median ± interquartile range as they did not pass normality test). The data suggest that Nav1.3 expressed by PMNs is responsible for the observed alteration of PMN transmigration. Nav1.3-dependent regulation of PMN
0 20 40 60 80 100 120
transmigration of mouse PMNs through fEnd.5 cell layer (% of medium + fMLP)
fMLP fMLP TTX
fMLP ICA
fMLP PTX-2 p = 0.0063
p = 0.0133 p = 0.0096
0 20 40 60 80 100 120
transmigration of mouse PMNs through collagen I (% of medium + fMLP)
fMLP fMLP
TTX fMLP ICA
fMLP PTX-2 p = 0.0065
p = 0.0001 p < 0.0001
A B
C
0 20 40 60 80 100 120
transmigration of human PMNs through HUVEC cell layer (% of medium + fMLP)
fMLP fMLP TTX
fMLP ICA
fMLP PTX-2 p = 0.0016
p = 0.0062 p = 0.0031
blood PMNs and HUVECs. However, in comparison with the mouse system higher TTX concentration of 200 nM and a pre-incubation of TTX, ICA121431 and PTX-2 with human PMNs for 15 minutes prior to adding PMNs to HUVECs was necessary to achieve a reduction of transmigration to 56 ± 19 % by TTX, to 62 ± 22 % by ICA121431 and to 59 ± 22 % by PTX-2 (Fig. 4C). Taken together, these data strongly suggest a regulating role for Nav1.3 not only for adhesion, but also for transmigration in both human and mouse PMNs.
Blocking of Nav1.3 negatively affects chemotactic migration of mouse PMNs in vitro Next we investigated which aspects of directed migration towards a chemotactic gradient of fMLP like accumulated distance, velocity or direction are affected by blocking Nav1.3 channels on mouse PMNs. Trajectory plots of PMN migration pathways towards fMLP throughout the recording time of 20 minutes in ibidi µ-slides show a directed migration for fMLP only, whereas blocking Nav1.3 with TTX, ICA121431 and PTX resulted in a non-directed migration pattern (Fig. 5A).
By measuring the Euclidean distance we could demonstrate that the shortest distance between the start- and endpoint of migration was significantly reduced for all three Nav1.3 channel blockers. Using fMLP as a chemoattractant the Euclidean distance was shortened from 72 ± 39 µm in control solution to 21 ± 29 µm with TTX, 18 ± 17 µm with ICA121431 and 10 ± 9 µm with PTX-2 (Fig. 5B). The directness of migration towards fMLP resembling the ratio of Euclidean and accumulated distance was significantly reduced from 0.53 for fMLP to 0.19 with TTX, 0.14 with ICA121431 and 0.07 with PTX-2 (not shown). These data indicate that Nav1.3 is required for directing the migration of PMNs within the fMLP gradient. Nav1.3 dependent directionality of PMN movement was further confirmed by the forward migration indices FMI parallel and FMI perpendicular, where FMI parallel was significantly decreased from 0.43 ± 0.27 for fMLP alone to 0.10 ± 0.2 with TTX, 0.04 ± 0.16 with ICA121431 and 0.01 ± 0.05 with PTX-2 (Fig. 5C).
Fig. 5. Nav1.3 controls PMN chemotaxis. (A) Trajectory plots of the migration path of PMNs treated with the Nav1.3 blocker TTX, ICA121431 and PTX-2 within a fMLP gradient located at the left side. (B) Migration of PMNs is less directly as the shortest distance between start- and endpoint (Euclidean distance) is significantly reduced by blocking Nav1.3 with TTX, ICA121431 and PTX-2 (n = 60; 3 repeated measurements of 20 cells each). (C) Direction of migration determined by the parallel forward migration index is not targeted towards the chemoattractant fMLP, if Nav1.3 is blocked by TTX, ICA121431 or PTX-2 (n = 60; 3x 20 cells). (D) Interestingly, only TTX reduced the accumulated distance and the velocity of PMNs (E) in a fMLP gradient, whereas addition of ICA121431 or PTX-2 showed no significant effect (n = 60; 3x 20 cells).
As FMI parallel exceeds FMI perpendicular and the Rayleigh test for homogenous distribution revealed a p-value < 0.05, only migration of PMNs towards fMLP fulfill the criteria for chemotactic migration, whereas addition of TTX, ICA121431 or PTX-2 blocked chemotactic movement of PMNs towards fMLP. Surprisingly, the mean accumulated distance PMNs migrated as well as the velocity was significantly reduced only by the combination of fMLP and TTX (dacc: 106 ± 35 µm, velocity: 0.12 ± 0.04) in comparison to fMLP (dacc: 135 ± 29 µm, velocity: 0.16 ± 0.03). These data indeed
0 50 100 150 200
Accumulated distance(µm)
p < 0.0001 p = 0.8850
p > 0.9999
fMLP +TTX +ICA +PTX2 0.00
0.05 0.10 0.15 0.20
Velocity (µm/s)
fMLP +TTX +ICA +PTX2
p < 0.0001 p = 0.6121
p > 0.9999 0
20 40 60 80 100 120
Euclidean distance (µm)
p < 0.0001
fMLP +TTX +ICA +PTX2
p < 0.0001 p < 0.0001
0.0 0.2 0.4 0.6 0.8
parallel forward migration index
fMLP +TTX +ICA +PTX2
p < 0.0001 p < 0.0001
p < 0.0001
A
B C
D
fMLP fMLP + TTX fMLP + ICA fMLP + PTX2
0 50 100
-50
-100
-100 -50 0 50 100 -100 -50 0 50 100 -100 -50 0 50 100 -100 -50 0 50 100
E
well. In contrast, the more specific Nav1.3 blockers ICA121431 and PTX-2 caused a slight and not significant reduction of the mean accumulated distance (ICA121431:
128 ± 36 µm and PTX-2: 132 ± 35 µm) and of the velocity (ICA121431: 0.15 ± 0.04 and PTX-2: 0.15 ± 0.04) (Fig. 5D and E). Taken together, we were able to show that PMN movement towards a gradient of fMLP is negatively affected by VGSC blockade in vitro.
Nav1.3 dependent whole cell current measurements
An obvious interpretation of the in vitro adhesion, migration and chemotaxis data is that Nav1.3 seems to carry a relevant function in PMNs. In order to verify an inhibitory effect of TTX, ICA121431 and PTX-2 on Nav1.3, we performed whole-cell patch clamp recordings on HEK 293 cells stably expressing Nav1.3. As expected, we observed that 100 nM TTX, 1.0 µM ICA121431 and 0.5 µM PTX-2 effectively inhibited sodium inward currents in these cells (Fig. 6A-C).
We also asked if voltage-gated sodium currents could be recorded in isolated mouse and human PMNs attached to cell culture plastic or stimulated with inflammatory cytokines.
In whole-cell voltage clamp recordings randomly selected cultured PMNs however, we were not able to detect any voltage-gated sodium currents in PMNs (data not shown, n = 10 for both species). Being able to identify and sort a population of PMNs evidently expressing Nav1.3 following kidney ischemia, e.g. Nav1.3. in Ly6G high cells, we again employed whole-cell patch clamp recordings in order to determine whether or not PMNs which stain for Nav1.3 are able to produce voltage-activated sodium currents. However, the voltage-activated sodium currents of 15 examined PMNs which bound Nav1.3-beads were only minimally elevated above baseline recordings (Fig. 6D). Thus although PCR and pharmacological experiments suggest that VGSCs are functionally expressed in both mouse and human PMNs, our patch clamp experiments on PMNs from ischemic organs favour more the possibility that expression of VGSCs is not high enough to generate voltage-activated sodium currents from the same quality as recorded on Nav1.3 transfected HEK 293 cells (Fig. 6E). Alternatively, expression of Nav1.3 in neutrophils might obey a very tight time window when these cells are activated in vivo, so that functional expression is rapidly lost under culture conditions in vitro.
Fig. 6. Nav1.3 is blocked by TTX, PTX-2 and ICA121431, but does not generate large sodium currents in neutrophils. (A - C) Representative current traces from HEK 293 cells with a stable expression of rat Nav1.3. Cells were held at -100 mV. The effects of the blockers TTX (A, 100 nM, 99 ± 1 % block, n = 6), PTX-2 (B, 0.5 µM, 54 ± 3 % block, n = 3) and ICA121431 (C, 1 µM, 77 ± 8 % block, n = 4) were explored on inactivated Nav1.3 channels induced by a 100 ms long pre-pulse at -70 mV before the test-pulse to 0 mV was applied for induction of a sodium current. Currents were evoked at 0.1 Hz. (D) Typical current trace from a Nav1.3-bead-binding PMN derived from mice following kidney ischemia. PMNs were held at -100 mV and sodium currents were activated by stepwise depolarizing pulses ranging from -100 to +20 mV in steps of 10 mV (see insert). Note that PMNs generated only minimal inward currents. (E) Typical current trace from Nav1.3 overexpressing HEK 293 cell. Voltage-activated sodium currents were evoked as described under D for PMN cells.
Lidocaine is not affecting TTX, ICA or PTX-2 mediated reduction of PMN adhesion and transmigration
The unselective VGSC blocker lidocaine has been shown to reduce myocardial infarct size [15, 29] and to inhibit several neutrophil functions like transmigration through endothelial cells [12]. Therefore, we finally explored if lidocaine has an impact on Nav1.3-dependent PMN adhesion to endothelial cells and to transmigration on collagen- coated transwell filters. Lidocaine (100 µM) reduced adhesion of mouse PMNs to fEnd.5 cells to 60 ± 9 % compared to medium controls. Blockade of VGSCs with TTX (100 nM, 58 ± 8 %) or ICA121431 (1 µM, 55 ± 7 %) or PTX-2 (0.5 µM, 59 ± 7 %) in combination with lidocaine revealed no further reduction in adhesion (Fig. 7A, n = 10).
Transmigration rates were not affected by adding 10 µM lidocaine (data not shown), but
significantly reduced to 54 ± 8 % by 100 µM lidocaine (Fig. 7B, n = 9). Co-incubation of 100 µM lidocaine with TTX (100 nM, 55 ± 12 %) or ICA121431 (1 µM, 55 ± 11 %) or PTX-2 (0.5 µM, 59 ± 7 %) did not show any additive effects in reducing the number of transmigrated PMNs (Fig. 7B), thus suggesting that lidocaine and the VGSCs blockers TTX, ICA121431 and PTX-2 might operate via the same mechanisms.
Fig. 7. Lidocaine-dependent blocking of PMN adhesion and transmigration. (A) Adhesion of mouse PMNs to TNFα-activated fEnd.5 cells is significantly reduced by lidocaine (100 µM), but combined treatment of lidocaine and TTX or ICA121431 or PTX-2 resulted in no further reduction in adhesion (n = 10). (B) Lidocaine (100 µM) reduces PMN transmigration through collagen-coated transwell filter significantly compared to controls with fMLP in the bottom chamber (fMLP), but combined treatment of lidocaine and TTX or ICA121431 or PTX-2 resulted in no further reduction of transmigration towards fMLP (n = 9).
0 20 40 60 80 100 120
adhesion of mouse PMNs to fEnd.5 (percent of medium )
lidocaine lidocaine TTX
lidocaine ICA
lidocaine PTX-2 medium
p < 0.0001
p > 0.9999 p = 0.4118
p > 0.9999
0 20 40 60 80 100 120
transmigration of mouse PMNs through collagen I (percent of medium + fMLP)
fMLP fMLP
lidocaine TTX fMLP lidocaine
fMLP lidocaine PTX-2 fMLP
lidocaine ICA p < 0.0001
p > 0.9999 p > 0.9999
p > 0.9999
A
B
I.5. Discussion
In the present study we investigated expression and functional roles of VGSCs in PMNs and show that both mouse and human PMNs express mRNA for most known VGSC α-subunits. However, only Nav1.3 protein is found in mouse PMNs in vivo when residing in the infarcted myocardial area or in injured kidney tissue after ischemia and reperfusion suggesting a common function of Nav1.3 for recruited PMNs. Accordingly, both unselective VGSC inhibitors (e.g. TTX and lidocaine) and substances preferentially blocking Nav1.3 (e.g. ICA121431 and Pterinotoxin-2) strongly attenuated PMN adhesion, transmigration and chemotaxis in vitro. These data suggest that Nav1.3, and possibly also other VGSC α-subunits are required for proper function of PMNs and thus of the innate immune system. These novel findings indicate that VGSCs like Nav1.3 in PMNs might be relevant molecular targets for immunomodulation.
While there is no doubt that one of the main functional roles of VGSCs is to generate the upstroke of the action potential in excitable cells, there is also meanwhile little doubt about the notion that VGSCs also perform diverse noncanonical roles in several types of cells in mammals [3]. The expression and functional roles of different VGSC α-subunits have been described in various types of immune cells including monocytes, astrocytes, dendritic cells and lymphocytes [3-5, 10]. Similar to our data on the role of Nav1.3 in PMNs, different VGSC α-subunits in immune cells were frequently reported to be important for cell motility and migration. Nav1.5 seems to be the predominant subunit in macrophages where it was reported to regulate endosomal acidification, being involved in phagocytosis, but also to act as a pathogen sensor [4-6]. Furthermore, Nav1.5 is involved in migration and proliferation in astrocytes [7]. Nav1.4 on the other hand seems to regulate CD4-positive lymphocytes [9], and Nav1.7 was reported to regulate migration and cytokine responses of dendritic cells [10]. While an early immunohistological study reported on expression of Nav1.3 in astrocytes, the function role of Nav1.3 in these cells was not examined [30]. In contrast to many types of immune cells, the expression of VGSCs in granulocytes has not been described in previous reports. We report here that both mouse and human PMNs express multiple VGSC α-subunits, and that at least Nav1.3 seems to be relevant for proper function of PMNs at least in vitro.
In a translational perspective our data correlate very well with the established immunomodulatory property of the unselective sodium channel blocker lidocaine, and also with the general assumption that an inhibition of PMN function accounts for this property of lidocaine [12, 14, 31]. However, several previous reports describing that lidocaine and other local anesthetics abbreviate the activity of PMNs have postulated VGSC-independent mechanisms. To our knowledge however, none of these studies explored whether or not PMNs express VGSCs. One argument speaking against the involvement of VGSCs for the effects of lidocaine on PMNs was noted in an early report describing that an inhibition of the production of superoxide anions in PMNs by local anesthetics is not mimicked by TTX [32]. In fact, we could confirm and also extend on this notion. We found that TTX in the nanomolar range as well as ICA121431 and PTX-2 evoke a slight but not significant reduction in the generation of reactive oxygen species in phorbol ester stimulated PMNs (Supplement Fig. S4). Therefore, other mechanisms than VGSCs indeed seems to account for the effect of lidocaine on ROS production in PMNs. As Nav1.5, Nav1.8 and Nav1.9 are TTX-resistant and ICA121431 as well as PTX-2 not block all α-subunits however, one cannot completely rule out a role of VGSCs for ROS production in PMNs. Nevertheless, our data clearly indicate that TTX as well as ICA121431 and PTX-2 rather effectively inhibit adhesion, transmigration and chemotaxis of PMNs in vitro. We also observed that lidocaine mimicked the effects all three blockers, but that the combination of lidocaine with any of the three substances did not induce additive effects. These results indicate that TTX, ICA121431 and PTX-2 and lidocaine target the same mechanism (e.g. Nav1.3) on PMNs to regulate properties like adhesion, transmigration and chemotaxis.
The presented results of this report reveal some weakness when it comes to identify which subcellular functions and signaling pathways in PMNs depend on VGSCs, and this question urge for further studies on this topic. Regulation of adhesion, transmigration and chemotaxis of course include multiple possible roles for VGSCs in PMNs, making it rather challenging to identify specific mechanisms. Accordingly, despite the large number of published reports on noncanonical roles of VGSCS in different kinds of cells, it is still unclear through which mechanisms they regulate different cellular functions [3].
While we detected only mininmally elevated and no prominent sodium currents in PMNs evidently expressing Nav1.3, the majority of cell types in which noncanonical roles of
