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Division of Respiratory Medicine, Institute for Lung Health, University of Leicester, Leicester, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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50% of cells. In both cell types, the K+ current was
blocked reversibly by charybdotoxin, which along with its
electrophysiological properties suggests it is carried by a channel
similar to the intermediate conductance Ca2+-activated
K+ channel. Charybdotoxin did not consistently attenuate
histamine or leukotriene C4 release, indicating that the
Ca2+-activated K+ current may enhance, but is
not essential for, the release of these
mediators. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IgE-dependent activation of both human and rodent mast cells is characterized by an influx of extracellular Ca2+, which is essential for subsequent release of both preformed (granule-derived) mediators and newly generated autacoids and cytokines. However, flow of ions such as K+ and Cl- are likely to play an important role in activation responses because they regulate cell membrane potential and thus influence Ca2+ influx (3). For example, in T cells, specific inhibition of the voltage-dependent K+ channel Kv1.3 by the scorpion toxin margatoxin inhibits their proliferation, IL-2 secretion, and hence delayed-type hypersensitivity responses (4).
Several ion currents have been identified in rodent mast cells, but the function of most of these remains unclear. In both the rat basophilic leukemia (RBL)3 cell line (RBL-2H3) , a model of mucosal mast cells, and rat IL-3-dependent bone marrow-derived mast cells (BMMC), an inwardly rectifying K+ channel (Kir) is open when the cells are at rest (5, 6). This channel, which is considered to be Kir2.1 (7), induces a resting membrane potential of approximately -70 mV. Activation-dependent currents have been identified in response to various secretagogues, including a nonselective cation current carrying Ca2+ and Na+ (8), specific Ca2+ influx through store-operated calcium channels (SOCC) (9), and an outwardly rectifying Cl- conductance (10). Adenosine activates an outwardly rectifying K+ channel in a GTP-dependent and pertussis toxin-sensitive manner which may explain adenosine-potentiated IgE-dependent degranulation (11).
Despite these observations, there are important differences between rodent and human mast cells with respect to mediator content and secretory and pharmacological responsiveness; therefore, from a clinical perspective it is essential that studies be performed on human cells. We have recently identified a voltage-dependent Cl- current and Ca2+-activated Cl- and K+ currents in the human mast cell line HMC-1 (12), but these cells are immature and lack high-affinity IgE receptors and are therefore unsuitable for studying mechanisms of IgE-mediated mast cell degranulation. In this study, we describe for the first time ion currents present at rest and following IgE-dependent activation in human lung mast cells (HLMC) and primary human mast cells derived from progenitors in adult peripheral blood (human peripheral blood-derived mast cells (HPBDMC)). In addition, we have performed a preliminary investigation into the role of a calcium-activated K+ current (KCA) in IgE-dependent mast cell secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following were purchased: Stem cell factor (SCF), IL-6, IL-10 (R&D Systems, Abingdon, U.K.); charybdotoxin (ChTX), Histopaque 1077, 2-ME, EGTA (Sigma, Poole, Dorset, U.K.), human myeloma IgE (Calbiochem-Novabiochem, Nottingham, U.K.), sheep polyclonal antihuman IgE (Serotec, Kidlington, Oxford, U.K.), mouse IgG1 mAb YB5B8 (anti-CD117; Cambridge Bioscience, Cambridge, U.K.), sheep anti-mouse IgG1 Dynabeads (Dynal, Wirral, U.K.); histamine, S-adenosyl-L-[methyl-3H]methionine (Amersham Life Science, Little Chalfont, Buckshire, U.K.), RPMI 1640/Glutamax/HEPES, antibiotic/antimycotic solution, MEM nonessential amino acids, and FCS (Life Technologies, Paisley, U.K.). Rat kidney histamine methyltransferase was a generous gift from Dr. S Harper (AstraZeneca R&D Charnwood, Loughborough, U.K.).
HLMC purification
HLMC were dispersed from macroscopically normal lung obtained within 1 h of resection for lung cancer as described previously (13). Mast cells were purified using immunomagnetic affinity selection with antimouse IgG1 magnetic beads coated with the mouse anti-c-kit mAb YB5.B8 (13). Final mast cell purity was >99% and viability >97%.
Following purification, HLMC were cultured overnight on 1% BSA-coated plastic (to prevent adhesion) in RPMI 1640/Glutamax/HEPES containing antibiotic/antimycotic solution, nonessential amino acids, 10% FCS, and 10 ng/ml SCF. Cells were sensitized with human myeloma IgE (2.5 µg/ml) as required.
Primary mast cell culture from human adult peripheral blood
Mast cells were grown from progenitors in adult peripheral blood
using a modification of the method described by Saito et al.
(14) for human cord blood. Briefly, the mononuclear
fraction from 150 ml adult peripheral blood was isolated on Histopaque,
incubated for 1 h at 37°C to remove adherent cells, and then
cultured in RPMI 1640/HEPES containing 5% heat-inactivated
pooled human serum, SCF (100 ng/ml), IL-6 (50 ng/ml), and IL-10 (10
ng/ml). Half the medium was replaced with fresh medium every 7 days.
Mast cells cultured this way are functionally mature by 3 wk in culture
in terms of histamine, leukotriene C4
(LTC4), tryptase, and cytokine release in
response to IgE-dependent activation (Refs. 15, 16, 17, 18, 19 and our
unpublished data). After 6 wk of culture, 50% of cells are mast
cells based on metachromatic staining. For electrophysiological
recording and study of mediator release, the mast cell population after
6 wk was purified using immunomagnetic affinity selection as described
above for HLMC, providing a 100% pure population of mast cells. These
were used in experiments for another 4 wk.
Cell viability
Mast cell viability, monitored by exclusion of trypan blue, was >97% in all experiments.
Electrophysiology
The whole-cell variant of the patch clamp technique was used
(20). Patch pipettes were made from boro-silicate
fiber-containing glass (Clark Electromedical Instruments, Reading,
U.K.), and their tips were heat polished, resulting in resistances of
typically 4–6 M
. The standard pipette solution contained 140 mM
KCl, 2 mM MgCl2, 5 mM EGTA, and 10 mM HEPES (pH
7.3). The standard external solution contained 140 mM NaCl, 5 mM KCl, 2
mM CaCl2, 1 mM MgCl2, and
10 mM HEPES (pH 7.3). These and other solutions used are shown in Table I
. For recording, mast cells were placed in 35-mm dishes containing
standard external solution.
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Mast cell mediator release
For analysis of histamine and LTC4 release, 1–2 x 104 mast cells were warmed to 37°C in 50 µl of culture medium in duplicate, and an equal volume of culture medium containing sheep anti-human IgE at twice the final concentration was added. After a 45-min incubation at 37°C, 100 µl of ice-cold medium was added and the cells were centrifuged at 500 x g for 4 min to pellet the cells. The supernatant was decanted, and control cell pellets were lysed in sterile deionized water for measurement of total histamine content. ChTX was preincubated with the cells for 10 min before activation where appropriate.
Histamine and LTC4 assay
Histamine was measured by a sensitive radioenzymic assay based on the conversion of histamine to [3H]methylhistamine in the presence of the enzyme histamine-N-methyltransferase using S-adenosyl-L-[methyl-3H]methionine as the methyl donor (13, 21). Histamine secretion is expressed as a percentage of total cellular content (cell lysate plus spontaneous release) and is corrected for spontaneous release. LTC4 was measured by ELISA according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Uppsala, Sweden).
Data presentation and statistical analysis
Data are expressed as mean ± SEM unless otherwise stated. Differences between groups of data were explored using Student’s paired or unpaired t test (two tailed) as appropriate. A p < 0.05 was considered to be statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two-thirds of HPBDMC (44 of 64 cells from 3 donors) were electrically silent at rest, with resting membrane potential around 0 mV. Of the remaining cells, 17 expressed small outwardly rectifying currents which often underwent irreversible "rundown" within the first minute of achieving the whole-cell configuration and with reversal potential and resting membrane potential of 0 mV suggesting a probable Cl- conductance. Three cells demonstrated a K+ current that was otherwise seen to develop acutely after IgE-dependent activation but never spontaneously in control cells recorded for 10 min (see below).
IgE-dependent ion currents in HPBDMC
HPBDMC were consistent in their response to IgE-dependent
activation, and seals generally remained stabile during activation and
solution changes. In 28 of 33 cells from 3 donors recorded at 29°C
(extracellular solution E1, pipette solution I2, Table I
), there was an acute negative shift in
membrane potential from a baseline mean -4.4 ± 3.14 mV to
-43.6 ± 3.1 mV within 2 min of adding anti-IgE (1/1000
dilution of stock polyclonal IgG fraction which gives optimal histamine
release) to the bath (Fig. 1A;
p < 0.0001 for all 33 cells). This was associated with the
rapid development of a whole-cell current (3.5 ± 0.6 pA at
baseline, 28.6 ± 4.5 pA after anti-IgE at +30 mV,
p < 0.0001 for all 33 cells) which reached a peak
within 20 s of first appearing (Fig. 1B). The current
appeared immediately as voltage steps were applied, did not decay
during a 100-ms pulse (Fig. 1, C and D), and
demonstrated inward rectification from membrane potentials positive to
about +20 mV (Fig. 1, C and D). Reversal
potential of this whole-cell current always correlated very closely
with the measured membrane potential. This current was carried
predominantly by K+ ions as demonstrated by a
positive shift in reversal potential to 0 mV on switching from 5 mM
external K+ to 140 mM external
K+ (14 of 14 cells; solution E2), and under these
conditions was observed to exhibit weak inward rectification (Fig. 1E). The current was dependent on the presence of
extracellular Ca2+ since either switching to
Ca2+-free extracellular bath solution (solution
E3) or adding 5 mM EGTA to the bath eliminated the
K+ conductance and shifted reversal potential to
0 mV in a fully reversible manner (5 of 5 and 3 of 3 cells
respectively) (Fig. 1, F and G). This
Ca2+-activated K+ current
(KCA) persisted for up to 45 min of recording in
the whole-cell configuration with minimal rundown during this period.
When recording cells that had been activated with anti-IgE for up
to 30 min before achieving the whole-cell patch clamp configuration,
the same current was present immediately on achieving the whole-cell
configuration in 9 of 10 cells, indicating that this current did not
develop as an artifact of patch clamp recording. In 4 cells, the
whole-cell K+ current demonstrated more marked
inward rectification (e.g., Fig. 1B), which may have been
highlighted by the absence of a significant Cl-
current (see below).
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G). This was matched by a decrease in current from
23.9 ± 4.9 to 6.0 ± 1.2 pA at +30 mV
(p = 0.003 for all nine cells tested). In the
three other cells, in which the KCA was
relatively large and demonstrated more marked inward rectification,
ChTX had much less effect in two and no effect in one. Because complete
depolarization was always observed when extracellular
Ca2+ was removed, this suggests the presence of a
second ChTX-insensitive KCA channel contributing
to the hyperpolarizing potential. Apamin, a blocker of small
conductance KCAs had no effect on two cells, but
produced a marked and partially reversible block in a third cell (data
not shown), which lends some support to this suggestion. Adding ChTX to
the bath before adding anti-IgE prevented membrane
hyperpolarization, which then appeared on washout of the ChTX (data not
shown). Addition of 30 µM barium was without effect on the
K+ current, excluding a contribution from a
classical Kir channel, but 300 µM barium produced a marked block at
both positive and negative potentials and accompanying membrane
depolarization (n = 3; data not shown).
Following elimination of the dominant K+ current
with Ca2+-free extracellular solution, a smaller
outwardly rectifying current with a reversal potential of 0 mV
remained, suggesting the presence of either a
Cl- or mixed cation current (Fig. 2). Reducing the extracellular
Cl- concentration to 11 mM from 151 mM using a
Ca2+-free Na+
methanesulfonate solution (solution E5 ) produced
a decrease in outward current from 36.0 ± 3.4 to 27.5 ± 4.3
pA at +130 mV (n = 4, p = 0.006) and a
positive shift in reversal potential of 20.7 ± 3.0 pA
(p = 0.006; Fig. 2B), indicating the
presence of a Cl- conductance not activated by
Ca2+. This Cl- conductance
appeared immediately following voltage steps and did not inactivate
during 100-ms pulses (Fig. 2A).
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The majority of HLMC (58 of 91 cells, 14 donors) were electrically
silent at rest with no inward or outward current (Fig. 3A). Resting membrane
potential in these cells hovered around 0 mV, and for all cells studied
was a mean -4.4 ± 1.4 mV. In 8 of these donors, 23 of 75 cells
recorded expressed outwardly rectifying currents of varying amplitude
at rest with mean reversal potential of -7.4 ± 3.8 mV,
suggesting these were likely to be dominated by a
Cl- conductance in the solutions used (I1, 2,
3;E1). In 8 of these cells, the current only activated positive to
about +30 mV and resembled the resting whole-cell current we have
recently described in the leukemic HMC-1 human mast cell line
(12) both in terms of its slightly delayed activation and
whole-cell current-voltage relationship (Fig. 3, B and
C). Reducing the concentration of external
Cl- ions from 151 to 11 mM by switching to
solution containing Na+ methanesulfonate
(solution E4) reduced outward amplitude of this current by 33.3 ±
6.8% (n = 2) at a command potential of +130 mV,
supporting the presence of a dominant Cl-
conductance (due to activation at positive potentials, it was not
possible to demonstrate the predicted positive shift in reversal
potential in low Cl- solution) (Fig. 3C). Increasing extracellular K+
concentration to 140 mM in these cells was without effect (data not
shown). In the other 15 cells, the resting outwardly rectifying
whole-cell current appeared immediately following voltage steps and
resembled the activation-dependent Cl-
conductance described above for both HPBDMC and HLMC below (Fig. 3, D and E). In 9 of 51 cells from 6 donors, small
linear currents were present at rest with mean reversal potential of
-21.8 ± 3.8 mV, suggesting the presence of open
K+ channels. This was confirmed by demonstrating
an appropriate shift in reversal potential to 0 mV on switching to 140
mM K+ externally (n = 3; Fig. 3F). In control experiments, no cells developed currents
spontaneously within 15 min of achieving the whole-cell configuration
(n = 15).
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Due to the instability of membrane seals with increasing temperature, HLMC were activated with sheep anti-human IgE at 27°C after the whole-cell configuration had been obtained. Even then, the whole-cell configuration was readily lost either spontaneously or during solution changes. In 11 of 14 donors, 18 of 41 cells responded to anti-IgE with the development of an increased whole-cell conductance. Because of the difficulty changing solutions, the calcium dependency of these anti-IgE-induced currents was determined by buffering intracellular Ca2+ with EGTA.
In 7 of 14 cells recorded with 5 mM internal EGTA, an outwardly
rectifying whole-cell current developed slowly over 10 min following
activation with anti-IgE and appeared immediately following voltage
steps (Fig. 4, A and
B). Current increased from a baseline 7.8 ± 1.7 to
62.5 ± 13.3 pA at +130 mV. Reversal potential in these cells was
-5.7 ± 3.0 mV at baseline and -2.9 ± 2.1 mV after
anti-IgE (p = 0.51), suggesting a dominant
Cl- conductance. This was confirmed in 2 cells
by reducing the extracellular Cl- concentration
from 151 to 11 mM (solution E4; Fig. 4C). The appearance of
this Cl- current with 5 mM EGTA in the pipette
suggests that it is activated by second messengers or cell volume but
not by Ca2+. In 11 of 27 cells recorded with 0.2
mM internal EGTA or no internal EGTA, whole-cell current at +130 mV
increased from 34.4 ± 15.1 to 103.9 ± 29.1 pA, and membrane
potential shifted in a negative direction from -5.1 ± 6.2 mV at
baseline to -25.3 ± 4.3 mV following IgE-dependent activation
(Fig. 4D). When comparing the IgE-dependent change in
membrane potential between 5 mM internal EGTA (2.9 ± 4.1 mV) and
0.2 mM or no internal EGTA (-20.2 ± 5.2 mV), there was a highly
significant difference (p = 0.003). This
negative shift in membrane potential with minimal
Ca2+ buffering compared with potent
Ca2+ buffering suggests that
KCAs are also opened by IgE-dependent activation,
as seen with HPBDMC above. With no EGTA in the pipette, outwardly
rectifying whole-cell currents developed within 2 min of adding
anti-IgE and again appeared immediately following voltage steps
(Fig. 4, E and F), but did not show the inward
rectification that was seen with the HPBDMC, perhaps in part due to the
relatively large Cl- component of the whole-cell
current in HLMC.
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G). In 140
mM K+, the current demonstrated weak inward
rectification similar to that seen in the HPBDMC. Furthermore,
addition of 100 nM ChTX reduced current at +130 mV from 85 ± 25
to 21.8 ± 1.8 pA and produced a shift in reversal potential from
-45.5 ± 12.5 to -12.5 ± 7.5 mV (n = 2),
suggesting that the same KCAs are expressed as in
HPBDMC. Calcium ionophore-induced currents in HLMC and HPBDMC
To further examine the presence of
Ca2+-activated currents in HPBDMC and HLMC, cells
were incubated with the calcium ionophore A23187 (1 µM). Within 2 min
of adding A23187 to the bath solution, a robust outwardly rectifying
whole-cell current developed in all cells recorded when the patch
pipette contained 0.2 mM EGTA (HPBDMC, n = 4; HLMC,
n = 17; Fig. 5, A–D). No current developed when the pipette contained 5 mM
EGTA (HLMC, n = 4) or when the cells were bathed in
Ca2+-free extracellular solution (HLMC,
n = 4), indicating that the Ca2+
ionophore-induced current was Ca2+ dependent.
In HLMC, current increased from a baseline 9.6 ± 1.9 to
179.8 ± 30.2 pA at +130 mV and in HPBDMC increased from a
baseline 13.7 ± 4.3 to 241 ± 71.2 pA. Interestingly, in
both cell types, this current had different characteristics to the
IgE-dependent currents described above in that it activated slowly,
suggesting it was carried by a distinct set of channels (Fig. 5, A and C). Mean reversal potentials for the
currents in HLMC and HPBDMC were -2.9 ± 3.4 and -4.0 ±
9.5 mV, respectively, suggesting the presence of a dominant
Cl- conductance. This was confirmed in ion
substitution experiments with current at +130 mV falling from 90
± 20 to 31 ± 7 pA and reversal potential shifting from
-7.5 ± 2.5 to 30 ± 10 mV following replacement of
extracellular Cl- with methanesulfonate (HLMC,
n = 2) (Fig. 5E). In contrast, increasing
extracellular K+ was without effect (HLMC,
n = 2; data not shown).
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To study the role of KCA activation in HLMC
and HPBDMC secretion, cells were activated with anti-IgE (1/1000
dilution ) in the presence of ChTX (1, 10, 100 nM). Mean net
histamine release from HLMC from 10 donors was 11.7 ± 3.0%
(range, 0–26.6% ). In 6 of 7 experiments with >7% net
histamine release, ChTX produced a variable but dose-dependent
inhibition of this (range, 11.4–80.5% maximal inhibition; Fig. 6). Mean net histamine release from
HPBDMC from 3 donors was 45.3 ± 7.51.3%
(p = 0.05 compared with HLMC). In one
experiment with HPBDMC, 100 nM ChTX produced a 33% inhibition of
release, but had no effect in 2 additional experiments.
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). In one
experiment using HPBDMC, there was no inhibition of
LTC4 release (9.6 ng/106
cells) by ChTX. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In rodent mast cells, Ca2+, K+, Cl-, and Na+ conductances have been identified using patch clamp recording, although the role of these in cellular responses remains poorly defined. Interestingly, currents vary between different rodent mast cell phenotypes, which may explain in part mast cell functional heterogeneity (26). For example, the RBL cell line and rodent BMMC, which are considered to represent a mucosal mast cell phenotype, express a strong inwardly rectifying K+ current at rest which is probably carried by subtype Kir2.1 channels (7) and which sets a stable resting membrane potential close to the K+ reversal potential at about -70 mV. In contrast, rat peritoneal mast cells (RPMC), typical of connective tissue-type mast cells, are either electrically silent at rest or express an outwardly rectifying Cl- conductance (27). RPMC release their histamine explosively within 2 min of IgE-dependent activation (28) unlike rodent "mucosal" mast cells which secrete histamine in a linear fashion over 30 min (29). It is therefore of interest that HLMC, which degranulate rapidly with a t1/2 for histamine release of 2 min following IgE-dependent activation (30), but which are located at a mucosal surface, and the phenotypically similar HPBDMC (15, 16, 17) are very similar to RPMC in terms of the ion currents expressed at rest.
Following IgE-dependent activation, there was an acute negative shift in membrane potential due to the opening of KCAs. This was most striking in the HPBDMC but also evident in the HLMC. This is the first electrophysiological evidence of a KCA in a mast cell from any species although Ca2+-dependent K+ efflux has been observed in RBL cells using 86Rb+ as a tracer (31). The inward rectification in high extracellular K+ and significant block by ChTX suggest that this K+ current is carried predominantly by the intermediate conductance Ca2+-activated K+ channel hIKCA1 (hsKCA4/hSK4/hIK1) or a closely related as yet unidentified family member (22, 23, 24, 25). Expressed hIKCA1 clones from human placental, lymph node, and pancreatic cDNA libraries demonstrate electrophysiological features virtually identical to the currents we have observed in both types of human mast cell, as well as those described for other hemopoietic cells, including T cells (22, 32) and macrophage/monocytes (33). The negative shift in membrane potential resulting from opening of these channels will increase Ca2+ influx both by increasing the electrical driving force for Ca2+ entry, but perhaps more importantly by increasing the open probability of the SOCC. These latter channels that carry Ca2+ into the cell demonstrate inward rectification at negative potentials (9), and thus carry larger Ca2+ currents at negative potentials. This would therefore be predicted to increase mediator release and influence Ca2+-dependent gene transcription.
Indirect evidence supporting a prosecretory role for
KCA channel opening in mast cells is provided by
the observation that our HPBDMC, in which the KCA
current predominates, and blood-derived mast cells cultured by other
workers using similar methods, release 40% of total cellular
histamine (Refs. 15, 16 , and our unpublished
data), whereas HLMC, in which the KCA current is
less marked, show marked heterogeneity releasing 0–30% of total
cellular histamine. However, although ChTX produced a significant block
of the whole-cell current, the effect on histamine and
LTC4 release was clearly very variable in HLMC
and minimal in HPBDMC. There may be several explanations for this
observation. Although ChTX depolarized activated cells, membrane
potential after the addition of ChTX was still usually more negative
(approximately -10 mV) than the resting value preactivation (0 mV).
This would still provide some driving force for
Ca2+ influx although the magnitude of the
Ca2+ current would be predicted to be reduced.
This persisting polarization suggests that we may have incompletely
blocked the ChTX-sensitive channels, that some degradation of ChTX may
have taken place due to the release of mast cell proteases and reactive
oxygen species, particularly during mediator release experiments where
cell number is higher, or that a second type of
KCA channel resistant to ChTX is present. The
latter is plausible because ChTX had little effect on the
K+ current in some cells and apamin, a blocker of
the small conductance KCA channels hsKCA2 and 3,
produced reversible inhibition of the whole-cell current in one of
three cells tested. Thus, depolarization required to attenuate
secretion may not have been consistently achieved. Another
consideration is that KCA activity is more
important for distal responses such as
Ca2+-dependent gene transcription, because in T
cells, hIKCA1 blockade with ChTX inhibits cell proliferation and
IFN-
production (34). Finally, hIKCA1 also plays a role
in cell volume regulation in T cells (25), which is
therefore another potential role for the KCA
channel in degranulating mast cells.
In addition to the KCA current identified,
immunological activation in both HPBDMC and HLMC also opened at least
one Cl- channel which was not dependent on
Ca2+ influx. This current was isolated by
recording HPBDMC in Ca2+-free extracellular
recording solution and HLMC with high intracellular EGTA. It appeared
slowly over 10 min following activation, was outwardly rectifying, and
appeared immediately following voltage steps with no decay over a
100-ms pulse. A similar current of lower amplitude was present in some
cells at rest and may be carried by the same channels, but the current
never developed spontaneously after achieving the whole-cell
configuration, and in HPBDMC usually ran down rapidly if present at
baseline. This indicates that the current appeared as a result of cell
activation and not as an artifact of patch clamp recording. A similar
current is present in a proportion of rat peritoneal and BMMC at rest
(27) and also develops slowly in these and RBL cells after
IgE-dependent activation (10, 35). Interpreting the role
of this Cl- current during cell activation is
difficult because the intracellular Cl-
concentration, which varies widely between cells, is not known for
human mast cells. The physiological extracellular
Cl- concentration is 100 mM, so if the
intracellular Cl- concentration is in the region
of 30 mM as has been estimated for RPMC (36),
Cl- currents will contribute to membrane
polarization since reversal potential for Cl- at
these concentrations is about -40 mV, and this will theoretically
promote Ca2+ influx. In support of this, blockers
of rodent mast cell Cl- channels such as
5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and
4,4'-diisothiocyanato-2,2'-disulfonic acid (DIDS) attenuate histamine
secretion but only in the micromolar range (36, 37).
However, NPPB also inhibits Ca2+ influx through
SOCC (37), and although DIDS attenuates secretion and
blocks Cl- channels, it does not inhibit
Ag-induced 36Cl- uptake in
RPMC which occurs rapidly, while the appearance of
Cl- channel activity is delayed
(36). Furthermore, although sodium cromoglycate is a
potent blocker of the IgE-dependent Cl-
conductance in RBL cells (10), it is only a weak
antagonist of secretion from HLMC. Thus, the role of the delayed
Cl- current in rodents remains unclear.
Conversely, if the intracellular Cl-
concentration in HLMC is similar to the extracellular
Cl- concentration, then
Cl- channel opening will depolarize the cell and
antagonize Ca2+ influx. With this scenario, one
could hypothesize that since the appearance of this current is delayed
following activation, it actually represents a negative feedback
pathway to provide a brake to the secretory response.
Most cells express Cl- channels that are believed to be important for the regulation of cell volume. Two members of the voltage-dependent family of Cl- channels, namely, the inwardly rectifying channel ClC-2 and the outwardly rectifying channel ClC-3, are widely expressed in mammalian cells and activate in response to reduced extracellular osmolarity. Currents carried through ClC-3 have similar physiological characteristics to the Cl- currents expressed in HLMC and HPBDMC, suggesting that this channel may carry the IgE-dependent Cl- current. The primary role of this current may therefore be to regulate cell volume following activation, perhaps in concert with the KCA. Firm molecular identification of the channels present and selective inactivation, for example, with antisense oligodeoxynucleotides, will answer these questions.
A second type of Cl- current was activated by Ca2+ influx in both HPBDMC and HLMC following exposure to the calcium ionophore A23187. This current may also have contributed to the whole-cell current following IgE-dependent activation, but if so was masked by the K+ and delayed Cl- currents. The A23187-induced current was clearly different from the other currents in terms of its activation kinetics, indicating it is carried by a distinct set of channels, and was very similar to the Ca2+-activated Cl- current described previously in other cell types including human neutrophils (38). It is interesting that this current was dominant after A23187-induced activation, with little evidence of the KCA current, whereas the KCA current was more pronounced following IgE-dependent activation. To some extent the relatively large ionophore-induced current may have hidden the smaller KCA current, but the correct intracellular signals following IgE-dependent activation may also have a critical role in permitting mast cell KCA opening, as phosphorylation, for example, is known to affect KCA channel gating (39). Similar observations have been made in neutrophils which also express KCA channels, but develop a dominant Cl- current similar to the mast cell current in response to calcium ionophore (38).
In this study, we have used the whole-cell configuration of the patch clamp technique to analyze HPBDMC and HLMC ion currents. As the cell is dialyzed by the pipette solution, there is the potential for washout of important intracellular constituents such as cyclic nucleotides which may themselves modulate ion channel function. Thus, it is possible that these cells express further currents which have not been identified in this study. Further analysis using the perforated patch technique will help address this. In addition, recording was limited to a temperature of 27–29°C because of the instability of seals at greater temperatures. Because mast cell activation and Ca2+ influx are temperature dependent and maximal at 37°C (40, 41), it is likely that the magnitude of Ca2+-activated currents is attenuated under our recording conditions and the time course of channel activation slowed.
In summary, we have described for the first time ion currents present in functionally mature human mast cells. Interestingly there is little electrical activity at rest, but immediately after IgE-dependent activation there is opening of a KCA, likely to be hIKCA1, which results in membrane hyperpolarization, and this is followed by opening of a calcium-independent Cl- channel. Specific molecular identification and knockout of these channels will permit accurate assessment of their role in human mast cell biology. Finally, we need to study tissue mast cells recovered from patients with specific diseases such as asthma to understand the role of these and other ion channels in mast cell disease. Ultimately, this may lead to novel therapeutic strategies based on modulation of human mast cell ion channel activity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter Bradding, Department of Respiratory Medicine, Glenfield Hospital, Leicester, LE3 9QP, U.K. E-mail address: pbradding{at}hotmail.com
3 Abbreviations used in this paper: RBL, rat basophilic leukemia; HLMC, human lung mast cell; HPBDMC, human peripheral blood-derived mast cell; BMMC, bone marrow-derived mast cell; RPMC, rat peritoneal mast cell; KCA, Ca2+-activated K+ channel; SOCC, store-operated Ca2+ channels; hIKCA1, human intermediate conductance Ca2+-activated K+ channel; Kir, inwardly rectifying family of K+ channels; ChTX, charybdotoxin; LTC4, leukotriene C4; NPPB, 5-nitro-2-(3-phenylpropylamino)benzoic acid; DIDS, 4,4'-diisothiocyanato-2,2'-disulfonic acid; SCF, stem cell factor.
Received for publication January 13, 2001. Accepted for publication August 14, 2001.
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-hexosaminidase and -glucuronidase from purified rat serosal mast cells. J. Immunol. 123:1445.-hexosaminidase and leukotriene C4 from cultured bone marrow-derived rat mast cells. Immunology 67:414.[Medline]
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