Institute of Molecular Physiology, University of Sheffield, Sheffield S10 2TN, United Kingdom
Submitted 3 April 2003 ; accepted in final form 3 June 2003
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ABSTRACT |
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monocyte/macrophage; inflammation; phagocytosis
Kv1.3 is the Kv channel subtype found in cells of myeloid cell lineage including T and B lymphocytes, platelets, and megakaryocytes. In T cells, Kv1.3 is reported to be a primary regulator of cell activation, maintaining the resting membrane potential and counterbalancing potassium flux necessary to maintain Ca2+ influx (3, 24). Blockade of Kv1.3 attenuates the production of proinflammatory cytokines and proliferation in vitro (18, 22, 33). Additionally, Kv1.3 channels are considered to be an important therapeutic target for autoimmune disease such as multiple sclerosis, where selective Kv1.3 channel block slows the progression in experimental models of the disease (9).
Less is known regarding the expression of Kv channels in monocytes and macrophages. Voltage-gated potassium currents resembling Kv1.3 channels have been recorded from human blood monocyte-derived macrophages, mammalian macrophages, microglia, and monocyte-derived cell lines (10, 12, 13, 19, 20, 26, 28, 29). In hippocampal microglia, toxins selective for Kv channels prevent proliferation in vitro (20). Additionally, macrophage Kv channel expression is highly variable according to the state of cell activation. Cytokines, Toll receptor activation, and phorbol esters all upregulate the expression of Kv channels (27, 36). Kv channel expression has been shown to be strongly upregulated in resident alveolar macrophages compared with blood monocyte-derived macrophages (29), but little more is known regarding the functional role of the Kv channels in macrophages.
In this study we sought to identify the molecular identity and functional role of the Kv channel subtype expressed in primary macrophages of the human lung. Human alveolar macrophages reside within both the alveolar spaces and the lung parenchyma. They play a major role in the host defense system of the respiratory tract, mediating the release of proinflammatory (IL-1 and TNF-) and anti-inflammatory cytokines (IL-10) in response to invading pathogens. Kv channel expression has previously been documented by Nelson et al. (29), demonstrating high expression of an outwardly rectifying K+-selective conductance compared with blood monocyte-derived macrophages. In this study we used the whole cell patch clamp recording technique to pharmacologically characterize Kv currents and RT-PCR to examine expression of Kv channel mRNA. Furthermore, we investigated the possible functional consequences of channel activation using measurements of membrane potential, proinflammatory cytokine release, and a phagocytosis assay. Preliminary results have appeared in abstract form (25).
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MATERIALS AND METHODS |
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Isolation of pure macrophage population. Macroscopically normal lung tissue derived from lung resections from patients undergoing surgery for carcinoma were used in this study. All patients provided informed consent. Lung tissue was stripped of pleura and chopped finely with scissors in sterile phosphate-buffered saline (PBS). The chopped tissue was washed with PBS over 100-µm-diameter-pore nylon mesh, and the lung macrophages were collected in the wash. The macrophage-rich suspension was spun (1,200 rpm for 5 min), and the pellet was resuspended in penicillin (1,000 U/ml) and streptomycin (100 µg/ml) containing DMEM (DMEM-PS). Macrophages were first isolated by adherence to tissue culture plastic, and resulting adherent cells were washed with sterile PBS to remove nonadherent cells. The remaining adherent cells (macrophages and myofibroblasts) were stored in DMEM-PS. Adherent cells were removed by scraping. For single cell studies the cells were pelleted (1,200 rpm for 4 min), resuspended in serum-free DMEM, and plated directly onto glass coverslips.
For extraction of total RNA, a pure macrophage population was isolated from contaminating cells (primarily myofibroblasts) by fluorescence-activated cell sorting (FACS). Cells that adhered to tissue culture flasks were incubated in serum-free DMEM for 2 h at 37°C. We prepared zymosan-BODIPY conjugate by incubating equal amounts of zymosan-BODIPY conjugate with opsonizing reagent for 1 h at 37°C. The zymosan-BODIPY conjugate with opsonizing reagent was then pelleted by centrifugation (1,000 g, 5 min). The pellet was washed three times in cold PBS and resuspended in 300 µl of cold PBS. One hundred milliliters of this suspension were added to 15 ml of cold serum-free DMEM and allowed to adhere to cells at 4°C for 30 min, before being incubated for 1 h at 37°C to allow phagocytosis to take place. Cells were then washed in cold PBS and treated with 1:1, 000 diluted goat F(ab')2 anti-rabbit IgG (heavy and light chain) R-phycoerythrin conjugate for 30 min at 4°C. A fluorescence-activated cell sorter (Beckman Coulter, High Wycombe, Buckinghamshire, UK) was then used to separate those cells with the green fluorescence of the ingested BODIPY-zymosan complex from cells with the red fluorescence of surface-adherent IgG.
RT-PCR. Total RNA from FACS-sorted macrophages was extracted with TRIzol (Life Technologies, Invitrogen). The RNA samples were treated with DNase I (Ambion, Hunting-don, UK) to remove any chromosomal DNA contamination. cDNA was made from the total RNA with Superscript II RT kit (Life Technologies) with oligo(dT) primers as recommended. As a control, duplicate RNA samples were incubated under identical conditions but without RT. PCR was conducted using prealiquoted PCR master mix (ABgene; Epsom, Surrey, UK) in a thermocycler (Hybaid, Ashford, Middlesex, UK). The PCR cycle conditions were 94°C for 2 min, then 35 cycles of 94°C/30 s, 55°C/30 s, and 72°C/60 s, followed by one 72°C/5-min step. PCR products were separated by agarose gel (1%) electrophoresis. cDNA prepared from human brain RNA (Invitrogen) was used for positive controls to test primers.
Electrophysiological recording. Standard whole cell patch clamp recordings were made 12-72 h following plating of macrophages onto glass coverslips with a HEKA EPC10 amplifier and on-line compensation for series resistance and capacitance. All currents were analyzed using Pulsefit (HEKA Electronik). Patch pipettes (filled resistance 5-10 M) were pulled from borosilicate glass and filled with solution containing (in mM): 150 KCl, 10 HEPES, and 10 EGTA corrected to pH 7.2 with KOH. Normal external saline was (in mM): 145 NaCl, 5 KCl, 10 HEPES, 12 glucose, 2 CaCl2, and 1 MgCl2 brought to pH 7.3 with NaOH. Test solutions were applied using a gravity-driven fast flow system, RSC200 (Intracel). For ion selectivity experiments, extracellular KCl was replaced with increasing concentrations of NaCl. The osmolarities of all solutions ranged from 300 to 310 mOsmol/l. All recordings were performed at ambient room temperature (20-22°C).
ELISA assay for secretion of IL-1. Adherent alveolar macrophages were plated into a 24-well plate and treated for 4 h with endotoxin lipopolysaccharide (LPS, 10 µg/ml). Cells were washed twice with extracellular saline; then control solution, 2',3'-benzoyl-(4-benzoyl)-ATP (BzATP, 200 µM), or BzATP plus margatoxin (1 nM) was applied for 30 min. The solution was then collected and directly assayed for the presence of IL-1
. Sandwich ELISA plates were generated using antibodies obtained from Endogen (Loughborough, UK).
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RESULTS |
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Whole cell recordings. Mononuclear lung macrophages were distinguished from myofibroblasts on the basis of their size (15-25 µm diameter), ruffled plasma membrane, and abundant phagocytic granules easily visualized by engulfed environmental carbon particles that were often apparent. Internal carbon particles suggest that the macrophages are of alveolar origin rather than from the interstitial population. Depolarization from -100 to -40 mV or greater activated an outward current (Fig. 2) in >95% cells. The rate of activation increased steeply with depolarization, and at potentials greater than -20 mV the current began to inactivate within several tens of milliseconds (Fig. 2A). Cell capacitance was 32.6 ± 2.3 pF (n = 23), and the density of the outward current recorded at 0 mV varied from 1.4 to 65 pA/pF (mean 15.2 ± 3.4 pA/pF, n = 23). Assuming a reversal potential for the current of -100 mV (see below), we used the peak current to calculate the cell conductance (G) as a function of potential. The voltage dependence of activation was fitted by G/Gmax = 1/{1 + exp[-(V - V1/2)/k]}, where half-maximal activation (V1/2) was -18.3 ± 0.8 mV (n = 5) and the slope factor (k) was 9.4 ± 0.7 mV (n = 5, Fig. 2C). The values are similar to those reported previously by Nelson et al. (29) from human lung macrophages. The outward current declined strongly during a maintained depolarization (Fig. 2B). This inactivation was well fit by a single exponential and was nearly independent of membrane potential.
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The outward current activated by depolarization was potassium selective because the tail currents reversed at a potential that varied with the extracellular potassium concentration (Fig. 2, D and E). A pulse to 40 mV for 100 ms was followed by steps to a range of potentials (Fig. 2, D and E). The reversal potentials (Erev) were related to the extracellular potassium concentration ([K]o) according to Erev = 62 log [K]o/150.
Two compounds have been well characterized as blockers of Kv1.3 channels in other cells and in heterologous expression systems; these are the scorpion toxin margatoxin (1) and CP-339,818 (30). Margatoxin (30-1,000 pM) blocked the current (at 20 mV) in human alveolar macrophages with an IC50 of 162 ± 11 pM (n = 5; Fig. 3, A and B). CP-339,818 blocked most of the outward current (at 20 mV); with 1 µM, 31 ± 9% (n = 5) of the initial current remained. The voltage range over which outward currents begin to activate in macrophages (-40 to -30 mV, Fig. 2A) is close to the resting potential (-44 mV, Ref. 27). We therefore asked whether Kv1.3 contributed to the resting potential of the cells. The zero-current (resting) potential in our experiments was -34 ± 1.1 mV (n = 7). Margatoxin induced a slow depolarization of the membrane potential to -4.6 ± 1.1 mV (n = 5, Fig. 3C).
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Phagocytosis assay. We used margatoxin to test the effect of blocking Kv1.3 channel activity on Fc receptor-mediated phagocytosis. Phagocytic cells were detected using fluorescently conjugated IgG-coated zymosan particles. The percentage of macrophages in the total cell population was counted by flow cytometry. In control conditions, 37 ± 7.6% (n = 3) of cells were sorted as having ingested zymosan particles. Margatoxin (1 nM) added at the same time had no effect on this proportion (35 ± 8.3%, n = 3).
IL-1 release. Release of proinflammatory IL-1
was assayed by ELISA following preincubation with LPS for 12 h. Stimulation of P2X7 receptors with BzATP (200 µM for 30 min) evoked a release of IL-1
above background (control 459 ± 21 pg/ml, n = 3; BzATP 1,351 ± 39 pg/ml, n = 3; P < 0.0001; Fig. 3D). Margatoxin had no effect on the secretion of IL-1
elicited by stimulation of P2X7 receptors (1,247 ± 56 pg/ml, n = 3; P > 0.1; Fig. 3D) compared with control in the absence of toxin. Stimulation of P2X7 receptors in the presence of high extracellular potassium (150 mM) also had no effect on secretion of IL-1
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DISCUSSION |
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High expression of Kv1.3 channel was confirmed by RT-PCR from total macrophage RNA. To date, Kv1.3 channel mRNA expression has only been reported for phorbol ester-differentiated THP-1 cells (10) and culture rat microglia (20). A novel FACS-based double-labeling sort was designed to isolate a pure population of macrophages and total RNA extracted. An array of Kv channel primers were designed against 14 channel subtypes; however, only the primers for Kv1.3 yielded a product to the correct size confirming the expression of Kv1.3 channels and no other Kv channel subtype.
Of all the myeloid cells, Kv channels have been most comprehensively investigated in T lymphocytes. Kv channels are thought to be responsible for setting resting membrane potential and providing the driving force for calcium influx and subsequent cell activation. Indeed, blocking Kv channels in vivo proves to be anti-inflammatory and ameliorates the progression of experimental encephalitis, a chronic inflammatory autoimmune disease (2). In current clamp recordings, we found a resting membrane potential of -34 mV, which is comparable with previous whole cell recordings from human alveolar macrophage (-44 mV: Ref. 28) and potential-sensitive dye measurements from rat alveolar macrophages (-37 mV: Ref. 5). At these potentials, Nelson et al. (29) report that steady-state inactivation was incomplete (30%), suggesting that the Kv channel would respond to changes in membrane potential and contribute to the resting membrane potential of lung macrophages; this is consistent with our findings. On the other hand, macrophages have a prominent inward rectifier potassium conductance (13), and this may be the major contributor in conditions with less leakage conductance.
Increases in cytosolic calcium and sodium are concomitant with Fc receptor cross-linking and phagocytosis (11, 16, 25). Phagocytosis is the initiating phase of host defense and inflammation involving activation of phagocytic receptors, Fc, or complement, triggering a cascade of tyrosine phosphorylation, polymerization of F-actin, and contractile forces leading to internalization of particles. FcR1 cross-linking in U-937 cells leads to opening of a calcium-activated nonselective cation channel (TRPM4: Refs. 11, 21). Additionally, Fc
R-mediated phagocytosis triggers superoxide generation and leads to activation of a nonselective cation channel and membrane depolarization in peripheral mononuclear cells (16) and peritoneal macrophages (4). Recently, a current has been described in U-937 cells activated by intracellular
-NAD+-free acid and extracellular hydrogen peroxide, which may account for this current (15). The activation properties of this current correspond to those reported for TRPM2 channels (34). In this study we demonstrate that, although Kv channels contribute to the resting membrane potential and thus the driving force for cation entry, margatoxin had no obvious effect on Fc receptor-mediated phagocytosis. This suggests that activation of the nonselective cation current is a consequence of phagocytosis and Kv channel activity is not necessary to support phagocytosis. However, this FACS-based assay is limited in its ability to resolve total number of bioparticles per macrophage and may not detect more subtle modulation of phagocytosis.
Finally, the level of cytosolic potassium is proposed to play a critical role in the activation of caspase 1 and the release of 17-kDa IL-1. High extracellular K+ blocks the processing and release of 17-kDa IL-1
from microglia (32), whereas a potassium/proton ionophore (nigericin) promotes the release of 17-kDa IL-1
from mouse peritoneal macrophages and human monocytes (31, 32, 39). Knockout studies have demonstrated a central role of P2X7 receptors in the generation and release of 17-kDa IL-1
(37). Activation of P2X7 receptors, a nonselective cation channel, would lead to membrane depolarization, activation of Kv1.3 channels, and K+ efflux. Thus we investigated the consequence of Kv1.3 channel blockade on release of IL-1
. A maximal concentration of margatoxin was found to have no effect on the release of IL-1
detected by ELISA. It is clear from our studies that depolarization-evoked activation of Kv1.3 channels is not necessary for release of proinflammatory IL-1
. Interestingly, high extracellular K+ also has no effect on cytokine release, demonstrating no requirement for K+ efflux in the release mechanism.
The present experiments indicate that although Kv1.3 sets the membrane potential of human alveolar macrophages, it seems not to play a critical role either in their phagocytic or cytokine-secreting activities. Data from this investigation demonstrate that the strong expression of Kv1.3 channels in human macrophages and channel activity may be involved in other macrophage-specific activities, for example, superoxide generation or nitric oxide production. Interestingly, Kv1.3 channels have been shown to form complexes with 1-integrins and function in triggering T cell activation (23). Activation of the integrin proteins is rapid and plays a central role in enabling attachment of T cells or monocytes to the corresponding ligand on cell surfaces and extracellular matrices. Integrin receptor activation has been shown to modulate Kv channel activity in monocytes/macrophages (8, 9). It would be interesting to test the potential role of Kv channel in adhesion and chemotaxis of human alveolar macrophages.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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Present address for H. Chirakkal: Centre For Human Nutrition, Div. of Clinical Sciences, Coleridge House, Northern General Hospital, Sheffield S5 7AU, UK.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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