Voltage-Gated Nav1.7 Maintains the Membrane Potential and Regulates the Activation and Chemokine-Induced Migration of a This information is current as Monocyte-Derived Dendritic Cell Subset of September 24, 2021. Katalin Kis-Toth, Peter Hajdu, Ildiko Bacskai, Orsolya Szilagyi, Ferenc Papp, Attila Szanto, Edit Posta, Peter Gogolak, Gyorgy Panyi and Eva Rajnavolgyi J Immunol published online 29 June 2011 Downloaded from http://www.jimmunol.org/content/early/2011/06/29/jimmun ol.1003345 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2011/06/29/jimmunol.100334 Material 5.DC1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published June 29, 2011, doi:10.4049/jimmunol.1003345 The Journal of Immunology

Voltage-Gated Sodium Channel Nav1.7 Maintains the Membrane Potential and Regulates the Activation and Chemokine-Induced Migration of a Monocyte-Derived Dendritic Cell Subset

Katalin Kis-Toth,*,1 Peter Hajdu,†,1 Ildiko Bacskai,* Orsolya Szilagyi,† Ferenc Papp,†,‡ Attila Szanto,x Edit Posta,* Peter Gogolak,* Gyorgy Panyi,†,‡ and Eva Rajnavolgyi*

Expression of CD1a defines a human dendritic cell (DC) subset with unique functional activities. We aimed to study the expression of the Nav1.7 sodium channel and the functional consequences of its activity in CD1a2 and CD1a+ DC. Single-cell 2 +

electrophysiology (patch-clamp) and quantitative PCR experiments performed on sorted CD1a and CD1a immature DC (IDC) Downloaded from showed that the frequency of cells expressing Na+ current, current density, and the relative expression of the SCN9A encoding Nav1.7 were significantly higher in CD1a+ cells than in their CD1a2 counterparts. The activity of Nav1.7 results in a depolarized resting membrane potential (28.7 6 1.5 mV) in CD1a+ IDC as compared with CD1a2 cells lacking Nav1.7 (247 6 6.2 mV). Stimulation of DC by inflammatory signals or by increased intracellular Ca2+ levels resulted in reduced Nav1.7 expression. Silencing of the SCN9A gene shifted the membrane potential to a hyperpolarizing direction in CD1a+ IDC, resulting

in decreased cell migration, whereas pharmacological inhibition of Nav1.7 by tetrodotoxin sensitized the cells for activation http://www.jimmunol.org/ signals. Fine-tuning of IDC functions by a voltage-gated sodium channel emerges as a new regulatory mechanism modulating the migration and cytokine responses of these DC subsets. The Journal of Immunology, 2011, 187: 000–000.

endritic cells (DC) act as conductors of the immune replenishes various tissues with interstitial DC (7). In peripheral system through connecting the innate and adaptive arms tissues, DC also differentiate from blood monocytes driven by D of various immune responses (1). Conventional DC microenvironmental factors generated by pathogens, inflam- differentiate from a common progenitor of macrophages via two mation, Ags, or metabolites (8). In vitro differentiation of mono- independent pathways into CD11c+ blood precursors with or cytes to CD1a2 and CD1a+ DC requires GM-CSF to support cell without the expression of membrane CD1 molecules (2, 3). CD1a, survival and IL-4 driving cell differentiation (9). These DC subsets by guest on September 24, 2021 widely used as a cell surface marker of human DC, is a type 1 resemble immature tissue DC (IDC) that can be further activated membrane protein (4) that is stabilized by captured self- or by stimulatory signals. Previously, we have shown (10) that the pathogen-derived modified lipids (5) to activate CD1a-restricted CD1a2 and CD1a+ DC subsets exhibit different functional prop- T lymphocytes (6). CD11c+CD1a+ DC differentiate to Langerhans erties and are able to skew T cell polarization toward tolerogenic cells in epithelial surfaces, whereas the CD11c+CD1a2 subtype or immunogenic directions, respectively. Upon Ag encounter and concomitant danger signals, IDC un- dergo drastic phenotypic and functional changes and the transition *Department of Immunology, Medical and Health Science Center, University of Debrecen, Debrecen, H-4032 Hungary; †Department of Biophysics and Cell Biology, toward mature DC (MDC). Activated MDC migrate to draining Medical and Health Science Center, University of Debrecen, Debrecen, H-4032 lymph nodes where they act as potent professional APC that are ‡ Hungary; Cell Biology and Signaling Research Group of the Hungarian Academy able to prime and polarize naive Ag-specific T lymphocytes to of Sciences, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, H-4032 Hungary; and xDepartment of inflammatory or tolerogenic directions (11). Biochemistry and Molecular Biology, Medical and Health Science Center, University The presence of voltage-gated ion channels was first described in of Debrecen, Debrecen, H-4032 Hungary excitable cells, where they are traditionally involved in the electro- 1 K.K.-T. and P.H. contributed equally to this work. genesis of action potential (12). Later, several studies reported that Received for publication October 13, 2010. Accepted for publication May 26, 2011. ion channels are also expressed in nonexcitable cells, including This work was supported by Hungarian National Research Fund Grants OTKA NK- immune cells (lymphocytes, macrophages, monocytes, etc.) (13– 72937 (to E.R.) and OTKA CK 78179 (to G.P.) and Ta´rsadalmi Megu´jula´s Operatı´v 15). The voltage-gated potassium channels (VGPC) are key ele- Program Grants TA´ MOP-4.2.2-08/1/2008-0019, TA´ MOP-4.2.2-08/1/2008-0015, and TA´ MOP-4.2.1/B-09/1/KONV-2010-007. P.H. is supported by a Janos Bolyai fellow- ments of sustaining a negative membrane potential required for ship. efficient Ca2+ signaling in these cells. Changes in the expression of Address correspondence and reprint requests to Dr. Eva Rajnavolgyi, Department of VGPCs and other ion channels accompany immune cell differenti- Immunology, Medical and Health Science Center, University of Debrecen, 1 Egye- ation, and thus may have an impact on cellular functions. In a recent tem Square Debrecen, H-4032 Hungary. E-mail address: [email protected] study, Black et al. (16) described that microglia cells, which ex- The online version of this article contains supplemental material. clusively reside to the CNS, express multiple voltage-gated sodium Abbreviations used in this article: ChTx, charybdotoxin; DC, dendritic cell; EC, extracellular; IDC, immature DC; MDC, mature DC; MMP12, matrix metalloprotei- channels (VGSC), playing a role in cell migration and phagocytosis. nase 12; Q-PCR, quantitative PCR; siRNA, small interfering RNA; TTX, tetrodo- The role of sodium channels in the activation and phagocytosis of toxin; VGPC, voltage-gated ; VGSC, voltage-gated sodium microglia and macrophages in experimental autoimmune ence- channel. phalomyelitis and multiple sclerosis was described, and the anti- Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 inflammatory role of sodium channel blockers was suggested (17).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003345 2 FUNCTION OF Nav1.7 IN CD1a+ DCs

It has also been reported that various VGSC antagonists are able to specification, CD80 FITC (Biolegend, San Diego CA), CD83 FITC influence the activation of lymphocytes; modify volume regulation, (Immunotech, Marseille, France), and CD86 PE (R&D Systems, Minne- migration, and apoptosis in Jurkat cells; and impair phagocytosis apolis, MN) for DC maturation as compared with isotype-matched control Abs (BD Pharmingen, San Diego, CA). Fluorescence intensities were and inflammatory responses of macrophages (18). However, the measured and analyzed by a FACSCalibur flow cytometer (BD Biosciences function of VGSC in immune cells is still elusive. Immunocytometry Systems, Franklin Lakes, NJ). CD1a+ and CD1a2 DC In our previous study, we characterized a novel VGSC, the Nav1.7 for quantitative PCR (Q-PCR) experiments were separated using a FACS channel in monocyte-derived DC, and demonstrated a developmental DiVa high-speed cell sorter (BD Biosciences Immunocytometry Systems). switch of Nav1.7 to VGPC Kv1.3 expression in the course of in vitro Q-PCR DC maturation (19). The a-subunit of Nav1.7 was shown to amplify Q-PCR was performed, as described previously (27). Briefly, total RNA small depolarization events to generate threshold currents close was isolated from sorted CD1a+ and CD1a2 DC by TRIzol reagent to the resting potential of neuronal cells (20). The SCN9A gene (Invitrogen, Carlsbad, CA). Reverse transcription was performed at 37˚C encoding for the Nav1.7 protein is expressed preferentially in no- for 120 min from 100 ng total RNA using the High Capacity cDNA Ar- ciceptive dorsal root ganglion neurons and in sympathetic ganglia chive Kit (Applied Biosystems, Foster City, CA). Q-PCR for Nav1.7, IKCa1, and MMP12 was performed with ABI PRISM 7900 (Ap- (21, 22). Nav1.7 expression is upregulated in pathophysiological plied Biosystems) with 40 cycles at 94˚C for 12 s and at 60˚C for 60 s conditions such as metastatic prostate cancer, where its activity using TaqMan assays (Applied Biosystems). All Q-PCR were run in potentiates numerous cellular activities of the metastatic cascade triplicates with a control reaction containing no reverse-transcriptase en- (23). Other studies also indicated that Nav1.7, via mutation or al- zyme. The comparative cycle threshold method was used to quantify transcripts relative to the endogenous housekeeping control gene 36B4. tered transcription, plays a key role in pain induction. Upregulation of Nav1.7 expression and activity results in increased pain sensa- Transfection of small interfering RNA Downloaded from tion, and therapies based on blocking Nav1.7 activity or silencing The mix of three different constructs of Nav1.7-specific and control small gene expression emerge as means for effective pain relief (24). interfering RNA (siRNA; Applied Biosystems) was transfected into dif- Consistent with this scenario, mice lacking the SCN9A gene through ferentiating IDC on day 3 at a final concentration of 1 pmol using the specific deletion in nociceptive sensory neurons exhibited reduced GenePulser X Cell electroporator and 0.4-cm cuvettes (Bio-Rad Labora- tories, Hercules, CA). After 2 d of transfection, the level of Nav1.7 mRNA responses to inflammatory, mechanical, and thermal pain (25). expression was tested by Q-PCR. Viability of the transfected cells was

Furthermore, mutations of the SCN9A gene that cause excessive controlled by 7-aminoactinomycin D and annexin V staining (BD Phar- http://www.jimmunol.org/ channel activities revealed the inherited pain syndrome eryth- mingen). ermalgia and paroxysmal extreme pain disorder (26). Despite the Electrophysiology potential therapeutic importance of Nav1.7 as a pain modulator, Patch clamping. Standard whole-cell patch-clamp techniques were used the major factors, which regulate the expression of SCN9A, have in voltage-clamp (current detection) or current-clamp (membrane potential not been identified. Moreover, the role of VGSCs and in particular measurement) configuration, as described previously (28). Whole-cell that of Nav1.7 in the regulation of Na+ flux to immune cells and measurements were carried out using Multiclamp 700B and Axopatch- their contribution to cellular functions are still poorly understood. 200A amplifiers connected to personal computers using Axon Instru- ments Digidata 1440 or 1320 data acquisition boards (Axon Instruments, In this study, we describe that the expression and function of the Foster City, CA). For data acquisition and analysis, the pClamp9 and 10 Nav1.7 channel are restricted to a subset of human monocyte- software packages (Molecular Devices, Sunnyvale, CA) were used. Pip- by guest on September 24, 2021 derived IDC specified by the expression of CD1a. We also identified ettes were pulled from GC 150 F-15 borosilicate glass capillaries (Clark the role of Nav1.7 in setting the membrane potential of CD1a+ IDC Biomedical Instruments, Pangboume, U.K.) in five stages and fire polished and thus modulating the threshold of DC activation. Our results to gain electrodes of 2-MV resistance in the bath. Series resistance com- pensation up to 85% was used to minimize voltage errors and achieve good show that the presence of active Nav1.7 channels in the cell voltage-clamp conditions (V , 5mV). CD1a+ and CD1a2 cells were + err membrane of CD1a IDC keeps the membrane potential at a depo- identified and selected for patch-clamp recording by anti-CD1a FITC la- larized state, thus protecting the cell from unnecessary or harmful beling in a Nikon TE2000 fluorescence microscope. When the SCN9 gene activation below an actively set threshold. Because the transition in differentiating DC was silenced by specific siRNA on day 3 of differ- entiation, the cells were labeled with anti-CD1a FITC Ab on day 5 and of IDC to MDC is a crucial decision step in triggering both used for the electrophysiological measurements. The same procedure was innate and adaptive immunity, these findings provide a new regu- applied for transfection of the control siRNA. latory mechanism to control DC functions. Moreover, differences Solutions. The normal bath or extracellular (EC) solution given in mM was in the expression level of the Nav1.7 channel may shed light as follows: 145 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 5.5 glucose, and 10 on the distinct responses of CD1a2 and CD1a+ cells to various HEPES (pH 7.35, 305 mOsm). The Na+-free bath solution contained (in stimuli. mM) the following: 145 choline-Cl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 5.5 glu- cose, and 10 HEPES (pH 7.35, 305 mOsm). The high K+ bath solution consisted of (in mM) the following: 150 KCl, 1 MgCl2, 2.5 CaCl2, 5.5 Materials and Methods glucose, and 10 HEPES (pH 7.35, 305 mOsm). The pipette solution for DC and cell cultures Nav1.7 measurements in IDC (in mM) is as follows: 140 KF, 5 mM NaCl, 11 K EGTA, 1 CaCl , 2 MgCl , and 10 HEPES (pH 7.20, ∼295 mOsm). + 2 2 2 Human monocyte-derived DC were generated from CD14 blood mono- The pipette filling solution for membrane potential recording in perforated cytes, as described previously (10). Activation of IDC was induced on day patch mode contained (in mM) the following: 150 KCl, 2 MgCl2, 1 CaCl2, 5 by inflammatory cytokine mixture containing 10 ng/ml TNF-a, 5 ng/ml 5 HEPES, 10 EGTA (pH 7.2), and 0.3 g/l Nystatin. For IKCa1 current IL-1b, 20 ng/ml IL-6, 75 ng/ml GM-CSF (PeproTech EC), and 1 mg/ml measurement (in mM), 150 KCl, 5 HEPES, 11 EGTA, 8.7 CaCl2, and 2 2+ PGE2 (Sigma-Aldrich, St. Louis, MO) for 24 h. When indicated, IDC were MgCl , (pH 7.2) were used in the pipette (free Ca concentration is ∼1 2+ 2 treated on day 5 with 180 ng/ml Ca ionophore (ionomycin; Sigma- mM). Aldrich) or with 250 nM thapsigargin (Calbiochem, La Jolla, CA) for 24 Determination of IKCa1 channel numbers. The whole-cell current was h in the absence or presence of EGTA that was added to the cultures at 5 evoked by voltage ramps, and the magnitude of the current was measured at mM together with 10 mM MgCl2 1 h before ionomycin, thapsigargin, or 285 mV (I285) and at 2120 mV (I2120). Because the reversal potential for tetrodotoxin (TTX) treatments. For the specific inhibition of Nav1.7 K+ is approximately 285 mV (E ), calculated from the composition of the m K channel activity, TTX (Alomone Laboratories) was used at 10–40 M pipette and the normal bath solution using the Nernst equation, the current concentrations for 24 h. at this voltage is purely the leak current. In contrast, the current measured Flow cytometry and cell sorting at 2120 mV consists of the IKCa1 current and the leak. Because the leak current reverses at 0 mV and has linear current-voltage relationship, its Phenotypic characterization of DC was performed by flow cytometry using value can be calculated at 2120 mV using the following formula: Ileak(@ fluorochrome-conjugated Abs, as follows: anti-CD1a PE for subtype 2 120 mV) = 2120/285 3 I285, and can be subtracted from the whole- The Journal of Immunology 3 cell current measured at 2120 mV to give the pure IKCa1 current at that maturation (Fig. 1C). Kinetic studies revealed the rapid upregu- 2 voltage [IIKCa1(@ 120 mV)]. Furthermore, the IKCa1 current does not lation (Fig. 2A) and downregulation (Fig. 2B) of Nav1.7 mRNA show time- and voltage-dependent activation (current-voltage relationship expression in the course of monocyte to IDC and IDC to MDC is linear); hence, the whole-cell conductance for IDC expressing only differentiation, respectively. As early as 3 h after the addition of IKCal channels can be calculated as follows: G = IIKCa1(@ 2 120 mV)/ (2120 mV 2 EK), where IIKCa1(@ 2 120 mV) is the current of IKCa1 GM-CSF and IL-4, the expression of Nav1.7 mRNA was upreg- 2 + channels at 120 mV and EK is the reversal for K . The single channel ulated and was increasing continuously during the entire process conductivity of IKCa1 was determined earlier (g = 11 pS) (29); thus, the of IDC differentiation (Fig. 2A), whereas DC maturation triggered number of channels in a cell is as follows: n =G/g. by an inflammatory cytokine mixture was able to downregulate Test substances. Toxins (TTX, charybdotoxin [ChTx]; Alomone Labora- tories) were dissolved in bath solution supplemented with 0.1 mg/ml BSA the expression of the Nav1.7 channels (Fig. 2B). Other stimuli, (Sigma-Aldrich) to prevent nonspecific binding of the toxins to the plastic known to induce DC activation [LPS + IFN-g, poly(I:C), CD40L] wall. TRAM-34 (Sigma-Aldrich) was dissolved in DMSO. Bath perfusion also led to the rapid reduction of Nav1.7 transcription (data not around the measured cell with different test solutions was achieved using shown). These results indicate that the role of the Nav1.7 channels a gravity-flow perfusion setup with six input lines and PE10 polyethylene is closely connected to the immature state of DC and acts pri- tube output tip having flanged aperture to reduce the turbulence of the flow. + Excess fluid was removed continuously. marily in the CD1a subset.

Cell migration assay The expression of Nav1.7 is downregulated via the elevation of intracellular Ca2+ concentration IDC were suspended in migration medium (0.5% BSA in RPMI 1640) at 106 cells/ml. Matrigel-coated or noncoated transmigration inserts (diameter 6.5 Although the full complexity of the molecular mechanism(s) that mm; pore size 3 mm) were obtained from BD Biosciences. MIP-1a che- regulates the activation of DC remains to be clarified, several Downloaded from mokine (PeproTech) was diluted in migration medium to 200 ng/ml and studies support the role of cytosolic Ca2+ signaling (reviewed in added to the lower chambers in a final volume of 600 ml. A total of 40 mM Ref. 30). Thus, next we investigated whether sustained high in- TTX or equal volume of DMSO was added to the lower and the equivalent 2+ upper chambers. When spontaneous trans-endothelial migration assays tracellular Ca concentration by itself would be able to decrease were performed, the migration medium in the lower chamber was RPMI the expression of the Nav1.7 channel. We found that elevation of 1640 plus 0.5% BSA in the absence of chemokines. IDC were added to the intracellular Ca2+ concentration, induced either by the Ca2+ ion- upper chamber in a final volume of 250 ml, and 24-h chemotaxis assays 2+ ophore ionomycin or by the sarco(endo)plasmic reticulum Ca - http://www.jimmunol.org/ were performed in 5% CO2 at 37˚C. At the end of the assay, the inserts were discarded and cells migrated to the lower chamber were collected. ATPase antagonist thapsigargin, was a potent inhibitor of Nav1.7 Migrated cell numbers were counted by using polystyrene standard beads gene expression and had a comparable inhibitory capacity to that (Sigma-Aldrich) by flow cytometry. Specific and control siRNA-trans- of the inflammatory cytokine mixture (Fig. 3A). A total of 5 mM fected IDC were prepared, as described above, and tested in the cell mig- extracellular EGTA partially or completely reversed the ion- ration assay. omycin- and thapsigargin-induced inhibition of Nav1.7 mRNA Statistical analysis expression, respectively. However, EGTA had marginal effect on the inhibition of Nav1.7 mRNA expression induced by the in- Statistical analysis and sample comparisons were made by unpaired t test, flammatory mixture. and the level of significance was set to 0.05. The mean and SEM of at least 2+ by guest on September 24, 2021 four or more independent experiments are shown. x2 test with Yates cor- The ability of increased intracellular Ca level to downregulate rection was used for correlation analysis. Nav1.7 channel expression was further verified by the decreased number of CD1a+ IDC expressing the Nav1.7 channel after 24-h thapsigargin treatment (Fig. 3B). Similarly to the inhibition of Results + mRNA expression, EGTA partially reversed the downregulation of Nav1.7 is expressed predominantly in the CD1a Nav1.7 expression in the plasma membrane. When we stimulated subpopulation of monocyte-derived IDC IDC by the inflammatory mixture, ionomycin or thapsigargin, and We have previously shown that functionally active Nav1.7 channels compared the cell surface expression of the activation molecules are expressed by monocyte-derived IDC and their expression is CD83, CD80, and CD86, we found that induction of CD83 and downregulated upon activation (19). In this study, we compared CD80 by ionomycin or thapsigargin, but not by the inflammatory Nav1.7 expression in the CD1a2 and CD1a+ IDC subsets pre- mixture could also be partially reversed by EGTA (Supplemental viously characterized by distinct functional activities. A robust Fig. 1). These signals also resulted in cytokine secretion in DC and inward, rapidly activating and inactivating Na+ current was de- production of TNF-a could be inhibited, whereas IL-6 secretion tected in CD1a+ IDC evoked by a 15-ms–long depolarization to was augmented by EGTA (Supplemental Fig. 1). These results 0 mV from a holding of 2120 mV upon perfusion with normal suggest that changes in calcium levels may have complex effects EC solution (Fig. 1A). The incidence of Na+ current detection in on DC activation and cytokine production. Hence, not only vari- CD1a2 IDC was significantly less frequent than in CD1a+ cells ous maturation stimuli of IDC, but environmental factors that (x2 test with Yates correction: CD1a+, n = 25 and CD1a2, n = 19, increase intracellular Ca2+ levels may trigger Nav1.7 channel p = 0.021). The Na+ current density of CD1a2 and CD1a+ IDC downregulation.

(CD, peak current at 0 mV divided by the capacitance of the cell, + a quantity proportional to the number of channels/unit membrane Nav1.7 dependence of the membrane potential in CD1a IDC area) also showed substantial differences, being significantly The role of VGSC in immune cells is unknown, but they are thought + higher in the CD1a subset (CDCD1a+= 283 6 7.6 pA/pF, to contribute to the maintenance of the resting membrane potential. CDCD1a2 = 243 6 13.4 pA/pF [n =9,p = 0.015]), as shown in Hence, we analyzed the membrane potential of Nav1.7-expressing Fig. 1B. However, the capacitance of the two subtypes was found CD1a+ IDC, their CD1a2 counterparts, and the corresponding to be the same (11.9 6 1.5 pF and 13.5 6 1.5 pF for CD1a+ and MDC. Whole-cell current-clamp measurements demonstrated that CD1a2 cells, respectively [n =9,p = 0.457]). Moreover, the the resting membrane potential of CD1a+ IDC is significantly relative expression of Nav1.7 mRNA measured by Q-PCR was depolarized (28.7 6 1.5 mV, n = 14) as compared with CD1a2 also higher in the immature CD1a+ than in the CD1a2 subset (fold IDC or MDC (between 240 and 260 mV, with an average of change CD1a+ versus CD1a2 = 3.5; p # 0.05) (Fig. 1C). The 247 6 6.2, n = 7, Fig. 4A). We postulated that this difference in expression of Nav1.7 was dramatically downregulated upon DC the membrane potential may originate from the presence of func- 4 FUNCTION OF Nav1.7 IN CD1a+ DCs

FIGURE 1. Expression levels of Nav1.7 in CD1a2 and CD1a+ IDC. A, Whole-cell Na+ current in a CD1a+ IDC recorded upon a 15- ms–long depolarization to 0 mV from a holding of 2120 mV. Arrows indicate the perfusion with normal and Na+-free extracellular sol- utions. B, Current density of Nav1.7 channels in IDC. The peak Na+ currents were determined at 0 mV and divided by the capacitance of the cell (measured in pF). This ratio was averaged for n . 6 CD1a+ and CD1a2 cells; error bars indi- cate SEM. C, Gene expression of Nav1.7 in monocytes (Mon.), IDC (open bars), and MDC (hatched bars). Triplicates of a typical experi- ment of three are shown. Error bars indicate SD.

*p , 0.05. Downloaded from

tional Na+ channels in CD1a+ IDC. To test this hypothesis, we de- cells did not change upon exposure to a Na+-free solution (Fig. http://www.jimmunol.org/ termined the extracellular Na+ sensitivity of the membrane po- 4D) similarly to the behavior of CD1a2 IDC lacking Nav1.7 tential in IDC. Fig. 4B demonstrates that the membrane potential channels (Fig. 4C). of CD1a+ IDC hyperpolarizes during transient exposure to a Na+- Fig. 4B–D shows that the membrane potential of IDC is sensitive free extracellular solution to values close to those recorded in to the extracellular K+ concentration: transient exposure of IDC to CD1a2 IDC and MDC of both CD1a2 and CD1a+ subtypes. This ahighK+ solution (150 mM K+ concentration) depolarized the solution completely abolishes the inward Na+ current in voltage- membrane potential to ∼0 mV. This and the relatively negative clamp experiments (Fig. 1A). On the contrary, the membrane membrane potential of CD1a2 IDC (Fig. 4A,4C) suggested the potential of CD1a2 IDC remained unchanged during the exposure presence of K+ channels in the membrane. As our previous study by guest on September 24, 2021 to a Na+-free extracellular solution (Fig. 4C). The Nav1.7 de- showed IDC lacking voltage-gated K+ channels (19), the expression pendence of the membrane potential was further studied by si- of a nonvoltage-gated K+ channel was postulated in the membrane lencing the Nav1.7 gene by using specific siRNA. Transfection of of IDC. Fig. 5A shows current traces recorded in CD1a2 IDC upon the Nav1.7-specific siRNA into IDC decreased the expression of a voltage-ramp protocol using a pipette solution containing 1 mM Nav1.7 mRNA by ∼60% (data not shown) and the number of free Ca2+. The biophysical and pharmacological properties of the Nav1.7 current-positive CD1a+ IDC to 13% (87% inhibition), current indicated the expression of IKCa1 Ca2+-activated K+ whereas 96% of CD1a+ cells treated by control siRNA remained positive for Na+ current expression (n $ 20 for control and SCN9A-silenced cells). Current-clamp recordings showed that the membrane potential of Nav1.7-specific siRNA-transfected CD1a+ IDC was significantly more negative (222.3 6 1.5 mV; n =8) than in nontransfected CD1a+ IDC (p , 0.001, Fig. 4A). Fur- thermore, the membrane potential of SCN9A siRNA-transfected

FIGURE 3. Effects of increased intracellular Ca2+ concentration on Nav1.7 gene expression in IDC. A, Nav1.7 gene expression in IDC (con- trol) and in IDC triggered by 250 nM thapsigargin, 180 ng/ml ionomycin, or inflammatory mixture was measured, as described in Materials and Methods, for 24 h in the absence (open bars) or presence (hatched bars) of

5 mM EGTA and 10 mM MgCl2. The expression level measured in MDC is shown as a positive control. B, Percentage of Nav1.7 expressing CD1a+ FIGURE 2. Transcriptional changes of Nav1.7 mRNA upon monocyte- IDC in control and in thapsigargin-treated cells in the absence (open bars) derived DC differentiation. A, Time course of Nav1.7 transcript levels in and presence (hatched bars) of EGTA. Cells were considered to express IDC upon addition of IL-4 and GM-CSF to monocytes. B, Nav1.7 mRNA Nav1.7 channels if a characteristic inward current was recorded at 0 mV levels during IDC to MDC transition induced by inflammatory cytokine test potential and the peak current exceeded 100 pA. Statistical calculation mixture given at the indicated time points. Triplicates of a typical exper- was based on the electrophysiological records from n . 13 cells in each iment of three are shown. Error bars indicate SD. group; error bars show SEM. The Journal of Immunology 5

FIGURE 4. Membrane potential of CD1a+ and CD1a2 IDC. Membrane potential of the cells was recorded in current-clamp configu- ration using the perforated patch method. The holding current was 0 pA. CD1a+ cells were identified in the patch-clamp microscope based on anti-CD1a FITC fluorescence stain- ing. A, Membrane potential of CD1a2 and CD1a+ IDC (open bars), the corresponding MDC (right-hatched bars), and Nav1.7 siRNA- transfected CD1a+ IDC (filled bar) measured in normal extracellular solution. Errors in- dicate SEM for n . 7 cells measured in each group. *p , 0.05. Membrane potential of a CD1a+ (B), a CD1a2 IDC (C), and a Nav1.7 siRNA-transfected CD1a+ IDC (D). Unless indicated otherwise, cells were perfused with normal extracellular solution. The switch of + the bath perfusion to a high K bath solution Downloaded from (right-hatched bar, 150 mM K+ concentration) or to a Na+-free bath solution (left-hatched bar) is indicated by the horizontal bars. The dashed lines indicate the zero potential. http://www.jimmunol.org/ channels: the current reversed at ∼280 mV and could be blocked posed that the functional role of Nav1.7 in DC may be connected by 25 nM ChTx (Kd value for IKCa1 is ∼5 nM) or with 125 nM to the maintenance of the immature state of the cells. We hy- + TRAM-34, a selective antagonist of IKCa1 channels (Kd ∼ 20–30 pothesized that CD1a IDC can get activated more readily than nM). The number of IKCa1 channels/cell was estimated for both CD1a2 cells and this sensitized state is connected to the CD1a2 and CD1a+ IDC, as detailed in Materials and Methods. activity. To test the potential role of Nav1.7 in fine-tuning CD1a+ No significant difference between the two DC subsets (p = 0.269) IDC activation, 10–40 mM TTX was used to inhibit Nav1.7 could be detected (Fig. 5B). The gene expression levels of IKCal channel function. Inhibition of Nav1.7 activity by TTX neither in the two IDC subtypes were also comparable (Fig. 5C). As both affected the monocyte to IDC and the IDC to MDC differentiation IDC subtypes express similar levels of IKCa1, we concluded that pathway nor modulated the viability or the internalizing capacity by guest on September 24, 2021 the lack of IKCa1 channels in CD1a+ cells cannot be responsible of IDC (data not shown). However, activation by optimal and for the depolarized resting membrane potential. suboptimal (10-fold dilution) concentration of the inflammatory

+ mixture for a short (6-h) activation period slightly increased the Nav1.7 regulates cytokine secretion and migration of CD1a level of CD83 expression (24 6 7% and 34 6 23% at optimal and IDC suboptimal concentrations, respectively; n = 5), but this effect was As the inflammatory nature of monocyte-derived MDC was at- statistically not significant (Fig. 6A). A similar sensitizing effect of tributed to the CD1a+ subset (10), and Nav1.7 channels were TTX treatment on DC could be shown when the production of predominantly expressed in this subpopulation of IDC, we pro- various DC-derived cytokines was tested after activation by the

FIGURE 5. Expression and activity of the IKCa1 channel in DC. A, Whole-cell current recorded upon a voltage-ramp protocol in a CD1a2 IDC in normal EC solution and in the presence of ChTx and TRAM-34. Dashed line represents zero current level. B, Number of IKCa1 channels on CD1a2 and CD1a+ IDC was calculated, as described in Materials and Meth- ods (n . 10); error bars indicate SEM. C, mRNA expression of IKCa1 channels in CD1a2 and CD1a+ IDC. Triplicates of a typical experiment of three are shown. Error bars indicate SD. 6 FUNCTION OF Nav1.7 IN CD1a+ DCs

FIGURE 6. Effect of TTX-induced Nav1.7 functional blockade on the activation of DC. IDC were treated with 40 mM TTX on days 2 and 5 of differentiation. Activation of IDC was induced by addition of the inflammatory mixture used at optimal or in 10-fold diluted concentra- tion on day 5, 1 h after TTX treatment. After 6 h, the excess of the mixture was removed by washing, and the activated MDC were cultured in fresh medium for another 16 h. A, The acti- vation status of MDC was monitored by mea- suring the expression of CD83 and by deter- mining the concentration of TNF-a (B), IL- 10 (C), IL-1b (D), and IL-6 (E) cytokines by Downloaded from ELISA. Results obtained from DC of a selected donor (88% of CD1a+ cells) of four are docu- mented. http://www.jimmunol.org/

cytokine mixture. In these experiments, IDC were treated with 40 measured. Furthermore, TTX was able to decrease both sponta- mM TTX on day 2 and in combination with the cytokine mixture neous (data not shown) and MIP-1a–induced migration of DC, on day 5. After 6-h activation, the excess of added cytokines was and the effects of TTX were more prominent in the Nav1.7- removed by washing the cells, the activated DC were cultured for expressing CD1a+ subpopulation (Fig. 7A). by guest on September 24, 2021 another 16 h in fresh medium, and the supernatants were subjected Silencing the Nav1.7 gene by a specific siRNA mixture in dif- to cytokine measurements. Although the absolute amounts of ferentiating IDC decreased spontaneous (data not shown) as well as cytokines varied among DC due to individually different CD1a+ MIP-1a–induced migration of IDC as compared with cells trans- ratios (10), a statistically significant increase of TNF-a (n =5;p = fected by control siRNA (Fig. 7B). Control experiments revealed 0.015; Fig. 6B) and IL-10 (n =5;p = 0.036; Fig. 6C) secretion was that transfection of siRNA did not induce CD83 expression, nor had detected in TTX-treated samples as compared with untreated an effect on cell viability. Concordant with the decreased migratory controls, respectively. This dramatic effect could not be detected potential of IDC with inhibited Nav1.7 channel expression and when IL-6 (Fig. 6D) or IL-1b (Fig. 6E) concentrations were function, relative gene expression of matrix metalloproteinase 12

FIGURE 7. Effects of the Nav1.7 channel blocker TTX and specific siRNA transfection on DC migration. Cell migration of IDC in- duced by MIP-1a was measured in Boyden chamber in IDC treated by 200 ng/ml TTX (A) or in Nav1.7-specific siRNA-transfected IDC (B). Mean + SD of three independent experi- ments is shown. Expression of MMP12 mRNA in control and TTX-treated (C) and control siRNA- and Nav1.7 siRNA-transfected IDC (D) was measured by Q-PCR. *p , 0.05. The Journal of Immunology 7

(MMP12), produced predominantly by CD1a+ cells (K. Kis-Toth, CD1a+ IDC results in a depolarized resting membrane potential. This unpublished observations) and involved in the release of tissue DC, conclusion is supported by the results showing the following: 1) was also downregulated upon TTX treatment (Fig. 7C) and in cells removal of extracellular Na+ hyperpolarizes the membrane of with silenced Nav1.7 gene expression (Fig. 7D). Nav1.7-expressing CD1a+ IDC, but does not affect CD1a2 IDC lacking Nav1.7 channels; 2) silencing of Nav1.7 gene expression in CD1a+ IDC results in a more negative and extracellular Na+-in- Discussion dependent resting membrane potential than in Nav1.7 expressing + The major function of IDC is to internalize exogenous soluble and CD1a IDC; and 3) the IKCa1/KCa3.1 channel is expressed in both particulate material and collect stimuli associated with environ- CD1a2 and CD1a+ IDC at similar levels. mental changes for delivering this molecular information to lymph Contribution of VGSC to set the resting membrane potential has node–resident T lymphocytes. Mobilization and maturation of DC been demonstrated in astrocytes (38, 39). Furthermore, they are also to highly efficient APC with the unique capability to prime Ag- implicated as key regulators of intracellular pH and cell migration specific T lymphocytes require inflammatory and danger signals. (40, 41). Our current-clamp experiments revealed that Nav1.7 Activation-induced changes of DC include the downregulation of channels are functional even at depolarized membrane potential; some endocytic/phagocytic receptors; upregulation of specific sets their presence is responsible for the maintenance of this depolar- of cytokine and chemokine receptors and adhesion and costimu- ized state, but they act exclusively in CD1a+ IDC. Although latory molecules; changes in morphology, cytoskeleton, and mo- depolarized membrane potential may induce inactivation of Na+ bility; and reorganization of the endolysosomal and MHC class II– channels, others reported that even at ∼0 mV an ample fraction of rich intracellular compartments (1, 31). Our previous electro- Nav1.7 channels is in available state (42, 43), and thus, the inward Downloaded from physiological and pharmacological analysis showed a characteris- (depolarizing) Na+ current may contribute to the regulation of tic change in the expression of ion channels associated with the membrane potential of CD1a+ IDC. Indeed, the lack of extracel- maturation of monocyte-derived DC. IDC were shown to express lular Na+, which abolishes the depolarizing Na+ influx through TTX-sensitive, rapidly inactivating voltage-gated Nav1.7 Na+ Nav1.7, hyperpolarizes the membrane potential in CD1a+ IDC. channels, whereas the dominant ion channel of MDC was the Alternatively, the lack of extracellular Na+ may interfere with Na+- + Kv1.3 K channel (19). dependent electrogenic transports, and thus, indirectly hyperpo- http://www.jimmunol.org/ The current study extends these findings by characterizing the larize the cells independent of Nav1.7 channels. In principle, the expression pattern and functional activity of Nav1.7 in distinct DC net inward current produced by the Na+/Ca2+ exchanger run in subsets generated from a common precursor. To our knowledge, forward mode could be responsible for extracellular Na+-dependent our results show for the first time that the frequency of cells membrane potential changes. We eliminated this possibility by our expressing Nav1.7 current and current density in Nav1.7-ex- siRNA experiment showing that knocking down Nav1.7 expression pressing cells is significantly higher in the CD1a+ DC pop- resulted in a hyperpolarizing shift in CD1a+ cells and the mem- ulation than in CD1a2 cells. This conclusion is based on ion brane potential became insensitive to extracellular Na+, similar to current measurement performed on individual cells identified by CD1a2 IDC with low Nav1.7 expression. These findings cannot be their CD1a expression in the cell membrane, the analysis of rel- explained by the sensitivity of the membrane potential to Na+/Ca2+ by guest on September 24, 2021 ative Nav1.7 mRNA expression measured in CD1a-sorted DC exchanger currents; rather, it can be interpreted as Nav1.7 de- subpopulations, and time dependence of Nav1.7 mRNA upregu- pendence of the membrane potential. These, in combination with lation and downregulation. the similar number of IKCa1 channels in the IDC subpopulations, Our data on the rapid activation-driven upregulation and support our conclusion that Nav1.7 channel is an active regulator of downregulation of the channel suggested direct transcriptional the membrane potential in CD1a+ IDC. control of Nav1.7 expression. Transcriptional regulation of ion As it was shown earlier, the existence of a negative expression has been reported in naive T cells, where the potential in various immune cells is necessary for the proper ac- expression of the Ca2+-activated K+ channel IKCa1 is increased tivation of Ca2+-dependent signaling (44). We propose that the upon activation (32). Elevation of intracellular Ca2+ concentration presence and activity of IKCa1 channels in both IDC subtypes in various cell types is one of the most rapid signal transduction may be responsible for maintaining the negative membrane po- events, and the importance, complexity, and impact of Ca2+-me- tential necessary for a sustained Ca2+ signaling during differen- diated signaling on the cell cycle of LPS-induced DC have re- tiation. The expression level of IKCa1 channels in CD1a+ and cently been described in mice (33). Furthermore, Ca2+ takes part CD1a2 IDC is much higher than in other immune cells, sug- in cell motility, which is one of the most characteristic features of gesting its exclusive role in the early phase of Ca2+-dependent DC DC. Various agents raising the intracellular concentration of Ca2+ maturation (45). have been investigated for their ability to alter the functions of The critical dependence of the membrane potential in Nav1.7 sodium channels in the plasma membrane and at the level of channels suggested that several physiological responses of CD1a+ mRNA expression (34–36). We report in this study the Ca2+-de- IDC may be impaired if Nav1.7 is inhibited. Although we per- pendent downregulation of Nav1.7 both at the mRNA and protein formed multiple functional studies to demonstrate the effect of levels in a subpopulation of human monocyte-derived DC, which TTX on IDC functions, we failed to detect inhibition of DC dif- may result in increased sensitivity to activation signals, as docu- ferentiation, phagocytosis, pinocytosis, or activation by the toxin mented by slightly increased CD83 expression and significantly (19). However, by using TTX and the siRNA strategy, we showed higher TNF-a and IL-10 secretion in the presence of TTX. Thus, that Nav1.7 takes part in regulating the secretion of certain cyto- the elevation of Ca2+ concentration, depending on the type and kines and spontaneous as well as chemokine-driven migration of strength of the activating stimulus (37), is able to modulate the IDC through or together with upregulating the MMP12 enzyme, expression and function of Nav1.7 channels that lead to changes known to be involved in DC mobilization from tissues. of the membrane potential. We propose that this process opens up In summary, we showed that Nav1.7 channels in IDC are ex- a DC state ready to respond to activation signals. pressed in the cell membrane in contrast to microglia, where they To reveal the functional role of Nav1.7 channel expression in are targeted to endosomes (16), and play an inevitable role in IDC, we demonstrated that expression of the Nav1.7 channel in the maintenance of the resting membrane potential, setting the 8 FUNCTION OF Nav1.7 IN CD1a+ DCs threshold and fine-tuning of DC activation and regulating the 17. Craner, M. J., T. G. Damarjian, S. Liu, B. C. Hains, A. C. Lo, J. A. Black, + J. Newcombe, M. L. Cuzner, and S. G. Waxman. 2005. Sodium channels con- migration of the human CD1a DC subset. We propose that the tribute to microglia/macrophage activation and function in EAE and MS. Glia depolarized membrane potential (∼210 mV) inhibits the activa- 49: 220–229. tion of highly inflammatory CD1a+ DC under a critical threshold, 18. 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