* Division of Respiratory Medicine, Institute for Lung Health, University of Leicester Medical School, United Kingdom;
Laboratory for Allergy Transcriptome, RIKEN Research Centre for Allergy and Immunology, National Research Institute for Child Health and Development, Tokyo, Japan; and
Department of Dermatology, University of Kyoto Faculty of Medicine, Japan
Correspondence: Dr. Peter Bradding, Department of Respiratory Medicine, Glenfield Hospital, Institute for Lung Health, University of Leicester Medical School, Groby Rd., Leicester, LE3 9QP, UK. E-mail: pbradding{at}hotmail.com
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Key Words: Ca2+ • Na+ • K+ • Cl- • gene array
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Persistent mast cell activation in disease may not only reflect continuing exposure to exogenous stimuli such as inhaled allergens but may have additional explanations such as activation by other inflammatory cell types and their products or intrinsic abnormalities in the signal transduction pathway for mediator release. Activation of human mast cells (HMC) is generally characterized by an influx of extracellular calcium (Ca2+), which is essential for subsequent release of preformed (granule-derived) mediators and newly generated cytokines [3 ]. The mechanism of sustained mediator secretion from mast cells in disease is therefore likely to include modulation of ion channels, which ultimately sets cellular resting membrane potential and carries Ca2+ fluxes required for intracellular signal transduction and secretory events. In this respect, flow of ions such as K+ and Cl- is likely to play an important role in activation responses, as they regulate cell membrane potential and thus influence Ca2+ influx [4 ]. For example, in T cells, specific inhibition of the voltage-dependent K+ channel (Kv)1.3 by the scorpion toxin margatoxin inhibits their proliferation, interleukin (IL)-2 secretion, and hence, in vivo delayed-type hypersensitivity responses [5 ].
Comparison of ion channel modulation in multiple excitation-secretion systems [e.g., in the central nervous system (CNS) and nonexcitable cell types] suggests that elevated secretory states in asthma could arise from aberrant high-affinity immunoglobulin E (IgE) receptor activation coupling to phosphomodulating enzymes acting at Ser/Thr or Tyr residues of resting channels; "constitutively active" second messenger/transducer protein systems simulating "noninactivating" (persistent) signals; or intrinsic abnormalities in ion channel (effector) function (or a combination of these). Irrespective of the mechanism(s) giving rise to "hypersecretory" states, we hypothesize that all mechanisms change the activity of the final effector ion channels involved in normal stimulus-secretion coupling. It is therefore important to identify these critical molecular effectors of HMC secretion and ultimately to study them in asthmatic versus normal populations.
Several studies have described ion currents in rodent mast cells in electrophysiological terms, but little attempt has been made to link these to cell function or identify the channels carrying them [6 ]. Even less is known about HMC ion currents, although we have identified resting and activation-dependent K+ and Cl- currents in the HMC-1 line and in human peripheral blood-derived (PBMC) and freshly isolated human lung mast cells (HLMC) [7 , 8 ]. However, expression of various currents/channels may not be readily evident using electrophysiological techniques, as ion channel recording is highly dependent on the in vitro conditions used. Molecular identification of these channels is critical if we are to be able to manipulate them to fully understand their role in mast cell physiology.
With the increasing number of cloned genes for different families of proteins including ion channels now identified, it is not feasible to undertake multiple polymerase chain reactions (PCRs) to identify all proteins, including channels potentially expressed by HMC. We have therefore undertaken high-density oligonucleotide probe arrays to study the expression of multiple genes in "resting" HLMC, skin mast cells, and human cord blood-derived mast cells (CBMC) at rest and after IgE-dependent activation in the presence or absence of dexamethasone. In this manuscript, we present and discuss the ion channel profile of these cells.
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Purification of human CD34+ cells
Mononuclear cells were separated from umbilical cord blood samples derived from healthy, nonatopic mothers (n=4) by density-gradient centrifugation using lymphocyte separation medium (Organon Teknika Corp., Durham, NC). The interface-containing mononuclear cells were collected. CD34+ cells were positively selected from cord blood-derived mononuclear cells using a CD34+ cell isolation kit and a magnetic separation column (MACS II, Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions.
Culture of HMC from CD34+ cells
Human CD34+ cells were suspended in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies, Gaithersburg, MD), supplemented with 1% insulin-transferrin-selenium-A supplement (Life Technologies), 50 µM 2-mercaptoethanol (Life Technologies), 100 units/ml penicillin (Life Technologies), 100 µg/ml streptomycin (Life Technologies), and 0.1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO; complete IMDM). CD34+ cells were cultured in the complete IMDM supplemented with 100 ng/ml stem cell factor (SCF; PeproTech EC Ltd., London, UK), 50 ng/ml IL-6 (PeproTech EC), and 2% fetal calf serum (Cansera, Rexdale, Canada) in 25- or 75-cm2 flasks (Iwaki Glass, Tokyo, Japan), as described elsewhere [9
]. After 11–14 weeks of culture, the cells (>99% tryptase-positive) were used for transcriptome and cytokine production assay.
Purification of HLMC
Macroscopically, normal human lung resected during surgery was obtained and processed after informed consent (n=2). HLMC were dispersed from chopped lung specimens by an enzymatic procedure and were purified by magnetic bead affinity selection using the antikit monoclonal antibody (Ab) YB5.B8 (BD PharMingen, San Diego, CA) as described previously [10
]. Counting, using a Neubauer hemocytometer after metachromatic staining with Kimura stain, was used to assess mast cell purity and numbers. The final purity of HLMC was >99%.
Skin mast cell purification and culture
Fresh samples of skin were obtained after breast reduction or mastectomy for breast cancer (n=3). Mast cells were enzymatically dispersed from human skin tissue and enriched as described previously [11
]. Percoll gradient-enriched cells were cultured with serum-free AIM-V medium (Life Technologies) supplemented with 100 ng/ml recombinant human SCF as described [11
]. After 4–5 weeks in culture, mast cell purity was >99%.
Activation of HMC
The HMC were sensitized with 1 µg/ml human myeloma IgE (a generous gift from Dr. Kimishige Ishizaka, La Jolla, CA) at 37°C for 48 h in the presence of IL-4 plus SCF and IL-6. After washing, the cells were suspended in the complete IMDM with the above cytokines. The cells were then challenged with 1.5 µg/ml rabbit anti-human IgE Ab (Dako, Glostrup, Denmark) or the culture medium alone at 37°C for 6 h. In some experiments, 10-6 M dexamethasone was added at the same time that anti-human IgE Ab was added.
GeneChip expression analysis
Human genome-wide gene expression was examined by using the Human Genome U133A probe array (GeneChip, Affymetrix, Santa Clara, CA), which contains the oligonucleotide probe set for 
22,000 full-length genes. This was performed in accordance with the manufacturer’s protocol (Expression Analysis Technical Manual) and previous reports [9
]. Total RNA (3–10 µg) was extracted from 107 mast cells of each phenotype and pooled (lung n=2 donors, skin n=3, cord blood n=4). Double-stranded cDNA was synthesized using a SuperScript Choice system (Life Technologies) and a T7-(dT)24 primer (Amersham Pharmacia Biotech, Buckinghamshire, UK). The cDNA was subjected to in vitro transcription in the presence of biotinylated nucleoside triphosphates using a BioArray high-yield RNA transcript labeling kit (Enzo Diagnostics, Farmingdale, NY). The biotinylated cRNA was hybridized with a probe array for 16 h at 45°C. After washing, the hybridized, biotinylated cRNA was stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR) and then scanned with a HP gene array scanner. The fluorescence intensity of each probe was quantified using a computer program, GeneChip Analysis Suite 4.0 (Affymetrix). The expression level of a single mRNA was determined as the average fluorescence intensity among the intensities obtained by 11 paired (perfect- matched and single nucleotide-mismatched) probes consisting of 25-mer oligonucleotides. If the intensities of mismatched probes were very high, gene expression was judged to be absent, even if a high average fluorescence was obtained with the GeneChip Analysis Suite 4.0 program. The level of gene expression was determined as the average difference (AD) using the GeneChip software. The percentages of the specific AD level versus the mean AD level of six probe sets for housekeeping genes [ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] were then calculated.
Data presentation
Gene expression data are presented as absent (-), present (% of GAPDH control), or marginal (M), as determined by the GeneChip software.
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K+ channels
K+ channels are widely expressed in many excitable cells (i.e., cells that conduct action potentials) and nonexcitable cells (i.e., cells that don’t conduct action potentials), such as cardiac tissue and leucocytes, respectively. The channel pore is very selective for K+ and is highly conserved across K+ channel families and throughout ontogeny. They can be gated by numerous mechanisms, including change in membrane voltage (voltage-dependent channels), binding of intracellular Ca2+ (Ca2+-activated channels), binding of second messengers (e.g., Kir1.0, 3.0, and 6.0 channels), and phosphorylation/dephosphorylation (e.g., Kir2.0 channels). Opening K+ channels moves the cell membrane potential toward the K+ equilibrium potential of about -80 mV and thus has a profound effect on cell membrane potential.
Inwardly rectifying K+ channels (Kir family; Table 1
)
The Kir family is comprised of seven subfamilies (Kir1.x–7.x) based on degree of similarity of their primary amino acid sequences [12
]. Each subfamily has distinct electrophysiological properties in terms of current rectification and channel gating. In spite of the apparently widespread expression of many of these K+ channel family members in many tissues, their role in mammalian physiology is uncertain. When present in cell plasma membranes, they often contribute to or dominate the resting membrane potential. In excitable cells, such as neurons or smooth muscle, this will have important effects in setting the threshold for cell activation. However, inwardly rectifying K+ currents have also been described in many nonexcitable cells, including epithelium, endothelium, and granulocytes [13
14
15
], where the setting of resting membrane potential may also influence stimulus-secretion coupling. Their expression therefore in human leucocytes may suggest important immunomodulatory roles. Human eosinophils and rodent mast cells express membrane currents at rest, consistent with the presence of Kir2 family members, and express mRNA for Kir2.1 [15
, 16
]. Using an RT-PCR-based, isoform-profiling strategy, we have recently described the expression of Kir family expression in HLMC from a single donor [17
]. In this patient, there was expression of Kir2.1–2.4, 3.1, 3.2, 6.1, and 6.2. In the current study, we have observed expression of Kir2.1 in skin mast cells, 2.4 in skin mast cells and HLMC, and 3.4 in HLMC. These results are therefore consistent with our previous observations in terms of Kir families expressed. Although we have never been able to record a Kir current in any HMC subset under basic electrophysiological recording conditions, these observations support the notion that pharmacological manipulation of the cells will be worthwhile to investigate the presence of these further. For example, Kir2.0 channels are closed by tyrosine phosphorylation and thus may be closed when normally cultured in the presence of SCF [18
], and G protein manipulation may open Kir3.0 channels. A further possibility is that these channels are located in intracellular membranes.
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Table 1. mRNA Expression (% GAPDH Control) for the Inwardly Rectifying (Kir) Family of K+ Channels and Ca2+-Activated K+ Channels (KCA) in HLMC, Skin, and CBMC
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There was no expression for sKCA1–3, but there was expression for the 
subunit of bKCA in skin mast cells and the ß4 subunit of bKCA, which attenuates channel sensitivity to charybdotoxin in all mast cell types.
Kv and two-pore K+ channels (Table 2)
Classical Kv channels of the Shaker (Kv1.x), Shaw (Kv2.x), Shab (Kv3.x), and Shal (Kv4.x) families are widely expressed throughout the CNS but are also evident in several nonexcitable cells including leukocytes [21
]. Perhaps the best-studied of these in terms of its role in immunology is Kv1.3, expressed by T cells, which when blocked, inhibits T cell mitogenic responses [5
]. In the mast cell types studied in this manuscript, we have not identified any Kv-like current electrophysiologically, and this, taken together with the absence of expression of any of these channels in the current study, suggests that Kv channels are not normally expressed by these mast cells. Similarly, we have never recorded a current characteristic of a two-pore K+ channel, and there was no expression for any of these.
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Table 2. mRNA Expression (% GAPDH Control) for Kv Families (Kv, KQT, ISK, and Eag-Related) and Two-Pore K+ Channels in HLMC, Skin, and CBMC
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subunits, which carry the Na+ current, and accessory ß subunits, which modify channel gating and serve as adhesion molecules mediating homophilic cell adhesion and adhesion to extracellular matrix proteins [23
, 24
]. Such channels have rarely been reported in nonexcitable cells, such as leukocytes [25
]. It is therefore of great interest that we have identified mRNA for the subunits Nav1.8 and the recently cloned SCN12A in all three types of mast cells studied, together with the ß1.1 subunit. We have not seen Navcurrents in HMC using the patch clamp technique but have not recorded cells under appropriate conditions (e.g., -80 mV holding potential to minimize depolarization-induced channel inactivation, internal caesium to block K+ currents, external methanesulfonate to minimize Cl- currents). It is interesting that T lymphocytes express a Nav and amiloride-sensitive Na+ channel, which cannot be recorded electrophysiologically under baseline conditions but appears within 30 min of coincubation of the cells with peptide-primed antigen presenting cells [26
]. This current in T cells appears to have a role in their proliferative response to antigen. As voltage-dependent channels tend to inactivate when held at positive potentials, Nav channels would not be expected to be open in resting HMC, which appear to maintain their resting membrane potential at approximately 0 mV [8
]. They would therefore unlikely be involved with the acute secretory response but could contribute to membrane depolarization when resetting the resting state or in other cell functions such as proliferation. The ß1.1 subunit could well play a role in mast cell adhesion and as mast cells are intimately related to peripheral nerves geographically within tissues, might provide the mechanism for their colocalization. |
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Table 3. mRNA Expression (% GAPDH Control) for Voltage-Dependent Na+ Channels (Nav), the Epithelial Na+Channel (ENaC), and the Brain Na+ Channel (BNaC2) in HLMC, Skin, and CBMC
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1 subunits were identified, which associate with a number of gating, modifying 2
, ß, and 
subunits [27
]. Again, their role in excitable tissue is easily understood, where membrane depolarization opens these channels permitting Ca2+ influx. A similar response in nonexcitable cells, such as mast cells, would be expected to produce degranulation, but these channels are generally thought not to be present in nonexcitable tissue. The finding in this study of expression of the subunit Cav3.3 in lung and possibly skin mast cells together with the 22 subunit in all types of mast cells is therefore intriguing. Again, we have not recorded these types of currents previously but have not specifically looked for them. |
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Table 4. mRNA Expression (% GAPDH Control) for Voltage-Dependent Ca+ (Cav) in HLMC, Skin, and CBMC
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Table 5. mRNA Expression (% GAPDH Control) for Nonselective Cation Channels of Purinergic Receptor (P2X), Transient Receptor Potential (TRP), Cyclic Nucleotide-Gated (CNG), and Hyperpolarization-Activated CNG (HCN) Families in HLMC, Skin, and CBMC
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TRP channels
The TRP family is the mammalian homologue to the Drosophila TRP channel gene [29
]. It is comprised of three subfamilies (TRPC, TRPM, and TRPV). Most of these allow passage of any cation, although in the presence of Ca2+, passage of other ions may be limited. Their role in cell function still remains uncertain, but several channels are activated by diacylglycerol or inositoltriphosphate and may thus link cell activation to Ca2+ influx. It has been proposed that TRPV6 is the Ca2+ release-activated current, which is opened in many cells by depletion of intracellular stores [34
], but this remains controversial [35
].
In the present study, we have observed expression of TRPC1 in human skin but not CBMC or HLMC. This is very interesting because of the marked heterogeneity evident among these mast cell phenotypes. CBMC and HLMC are very similar in terms of their mediator content and pharmacological responsiveness, whereas skin mast cells are quite distinct [36 , 37 ]. It is therefore possible that unique skin mast cell secretogogues, such as compound 48/80, codeine, and complement, could mediate their effects through activation of TRPC1, and the IgE-dependent pathway, which is common to all mast cell phenotypes, might signal via an alternative Ca2+ influx pathway such as TRPV6. Unfortunately, we were unable to examine for expression of TRPV6 in this study, as it was not represented on the gene chips used. In addition to TRPC1, we observed that all mast cells expressed TRPV2. This channel is activated by heat and growth factors [38 , 39 ] and so might mediate Ca2+ influx in response to SCF, for example, or the tissue response to thermal injury. CBMC and HLMC but not skin mast cells also expressed TRPM2, which could contribute to functional heterogeneity as well. This channel is activated by the second messenger adenosine 5'-diphosphate-ribose and oxidative stress and is thought to be involved in the respiratory burst in neutrophils [40 ].
Cyclic nucleotide-gated channels
There was no expression of classical or hyperpolarization-activated cyclic nucleotide channels in any of the mast cell phenotypes studied.
Cl- channels (Table 6)
Cl- channels are ubiquitous and present in most excitable and nonexcitable cells [41
]. In rat mast cells, it has been suggested that opening Cl- channels is prosecretory, although whether this is true remains unclear [6
]. In the HMC-1, we have described currents and mRNA expression, suggesting the presence of the ClC channels ClC3 and ClC5 [7
]. We have also shown that currents typical of ClC3 and ClC5 are also present in HLMC [8
]. In the present study, there was clear expression of ClC3 mRNA in all types of mast cells. It is interesting that ClC5 was expressed in CBMC but not skin or HLMC, suggesting that HLMC don’t express this channel, and the ClC5-like current is carried by a related but an unidentified channel or that mRNA copy number was below the limit of detection. In addition, we identified expression of ClC7 in CBMC and HLMC. These ClC channels are believed to have predominantly intracellular roles regulating ionic composition of intracellular organelles [41
], but ClC3 has been implicated in the regulation of cell volume [42
], and another current very similar to ClC5 has been implicated in the control of cell regulation in astrocytes [43
]. Knockdown studies in HMC will help define their exact role in mast cell function.
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Table 6. mRNA Expression (% GAPDH Control) for Voltage-Dependent Cl- (ClC Family), Ca2+-activated, and intracellular Cl- Channels (CLIC Family) in HLMC, Skin, and CBMC
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We have described previously the presence of a Ca2+-activated Cl- channel (ClCA) in HMC-1, HLMC, and CBMC. Many of these ClCA channels have not yet been cloned. One family that has been cloned is the ClCA family present in epithelia, but these were not expressed by any of the mast cells investigated.
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Received December 11, 2002; accepted January 27, 2003.
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