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Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1332-1340
Characterization of Functional Vanilloid Receptors Expressed by Mast
Cells
By
Tamás Bíró,
Marcus Maurer,
Shayan Modarres,
Nancy E. Lewin,
Chaya Brodie,
Géza Ács,
Péter Ács,
Ralf Paus, and
Peter M. Blumberg
From the Molecular Mechanisms of Tumor Promotion Section, Laboratory
of Cellular Carcinogenesis and Tumor Promotion, National Cancer
Institute, National Institutes of Health, Bethesda, MD; and the
Department of Dermatology, Charité, Humboldt Universität zu
Berlin, Berlin, Germany.
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ABSTRACT |
Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act
through specific vanilloid receptors on sensory neurons. The C-type
receptor is coupled to 45Ca uptake, whereas the R-type is
detectable by [3H]RTX binding. We describe here specific
vanilloid responses in murine mast cells (MCs). In the MC lines and in
bone marrow-derived mast cells, capsaicin and RTX induced
45Ca uptake similarly to that observed for cultured rat
dorsal root ganglion neurons (DRGs). This response was antagonized by
the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of MCs with capsaicin or RTX induced desensitization to subsequent stimulation of 45Ca uptake. The potency for desensitization
by RTX in the MCs corresponded to that for 45Ca uptake,
whereas in DRGs it occurred at significantly lower concentrations
corresponding to that for the high-affinity [3H]RTX
binding site. Consistent with this difference, in MCs we were unable to
detect [3H]RTX binding. Vanilloids were noncytotoxic to
the MCs, in contrast to the DRGs. Although vanilloids did not cause
degranulation in MCs, in the P815 clone capsaicin evoked selective
interleukin-4 release. We conclude that certain MCs possess vanilloid
receptors, but only the C-type that functions as a channel. Our finding
that MCs can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of inflammation in response to vanilloids.
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INTRODUCTION |
A SUBPOPULATION OF primary afferent
neurons, located in the dorsal root and trigeminal ganglia, can be
defined by their selective susceptibility to the effects of
capsaicin,1,2 the major pungent ingredient of hot peppers
of the plant genus Capsicum, and to its ultrapotent analog,
resiniferatoxin (RTX), a naturally occurring irritant tricyclic
diterpene3 that combines structural features of the phorbol
ester tumor promoters and of capsaicin.4 Although no exact
neurochemical correlation exists, these neurons in general contain
calcitonin-gene related peptide (CGRP) and tachykinins such as
substance P (SP).5
Upon activation, capsaicin-sensitive nerves both transmit signals to
the central nervous system and release neuropeptides such as SP and
CGRP in the periphery.6 The role of this latter, efferent
function of the sensory neurons is crucial in inducing neurogenic
inflammation, a process that can be best modeled by the application of
capsaicin and related vanilloids.7
Capsaicin and its analogs act via the stimulation of vanilloid
receptors on sensory neurons.8,9 The effects of capsaicin can be described as three consecutive phenomena: first, capsaicin excites neurons bearing the vanilloid receptors; it then desensitizes them to subsequent stimuli; and, finally, depending on its time of
action and concentration, it causes neurotoxicity.2 The mechanisms of excitation, desensitization, and neurotoxicity are distinct entities and individually represent the variety of processes elicited by vanilloids. Previous studies have identified two vanilloid receptor subclasses (classified as C- and R-type vanilloid receptors) on dorsal root ganglion neurons (DRGs),10
defined by distinct pharmacology and physiology and detected by
45Ca uptake and [3H]RTX binding assays,
respectively, suggesting that different vanilloid induced mechanisms
can be mediated by different receptor subclasses (Table
1).
The action of capsaicin in inducing neurogenic inflammation involves,
among other cell types,2 the activation of mast cells (MCs). This finding is generally attributed to two facts. First, capsaicin-sensitive nerves are in anatomical contact with MCs in a
variety of tissues.11,12 Second, neuropeptides released from sensory neurons by capsaicin can induce MC degranulation (resulting in the release of, eg, proteoglycans, histamine, and serotonin) as well as the production and release of a wide array of
proinflammatory cytokines including interleukins (ILs) and tumor
necrosis factor- (TNF-).13,14 These MC mediators then can further stimulate the release of SP and other peptides from sensory
nerves, and antidromic stimulation of these neurons can further induce
MC activation.15 Thus, this bidirectional MC-sensory neuron
autocatalytic loop can amplify the efferent function of sensory
neurons, eventually resulting in neurogenic inflammation.16
This hypothesis is supported by a number of reports showing that the
induction of neurogenic inflammation by capsaicin in vivo is associated
with MC activation. For example, capsaicin induced inflammation of the
skin17,18 and gastric mucosa19 have been
described to be accompanied by MC degranulation. Furthermore, the
inflammatory response to the in vivo administration of capsaicin is
reduced by inhibitors of MC degranulation or antagonists of MC products
histamine or serotonin.17,20
These actions of capsaicin on MCs have generally been considered to be
indirect via the release of SP and other peptides. We show here the
existence of functional capsaicin receptors on MC lines, the
stimulation of which results in the influx of calcium, desensitization
of mast cells to subsequent vanilloid stimulation, and production and
release of the proinflammatory cytokine IL-4. We suggest that, in
addition to its action on sensory neurons, capsaicin may also act
directly on MCs. Moreover, these findings represent the first example
of nonneuronal cell lines expressing vanilloid receptors. The presence
of the vanilloid receptor on MCs may help to clarify the complex in
vivo effects of vanilloids and, furthermore, expand the range of their
potential therapeutic applications. In addition, the identification of
cell lines containing C-type vanilloid receptors should greatly
facilitate the biochemical and molecular analysis of these
receptors.
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MATERIALS AND METHODS |
MC/9, P815, 10P-2, 10P-12, 11PO-1, and RBL-1 cells were purchased from
ATCC (Gaithersburg, MD). PT-18 and RBL-2H3 and CFTL-12 cells were
generous gifts from Drs J. Rivera and J. Pierce (National Institutes of
Health, Bethesda, MD), respectively. [3H]RTX
was synthesized by the Chemical Synthesis and Analysis Laboratory, NCI-FCRDC (Frederick, MD). 45Ca (CaCl2) and
[3H]5-hydroxytryptamine (serotonin) were purchased from
DuPont-New England Nuclear (Boston, MA). Nonradioactive RTX and
capsazepine were from LC Laboratories (Woburn, MA). Capsaicin and
adenosine-5-triphosphate were from Sigma Chemical Co (St Louis, MO).
Ruthenium red was purchased from Research Biochemicals International
(Natick, MA).
Cell cultures.
The following media and supplements (GIBCO BRL, Gaithersburg, MD) were
used for the culture of mast cell lines: Dulbecco's modified Eagle's
medium (DMEM) supplemented with 116 mg/mL L-arginine, 36 mg/mL
L-asparagine, 6 mg/mL folic acid, 0.1 mmol/L nonessential amino acids,
1 mmol/L sodium pyruvate, 4 mmol/L L-glutamine, 0.05 mmol/L
2-mercaptoethanol, 10% fetal bovine serum (FBS), and 45% conditioned
medium (T-Stim; rat growth factor with concanavalin-A; Collaborative
Biomedical Products, Bedford, MA) for MC/9 cells; RPMI-1640 medium
supplemented with 10% FBS, 20% T-Stim, 0.1 mmol/L nonessential amino
acids, 1 mmol/L sodium pyruvate, 4 mmol/L L-glutamine, and 0.05 mmol/L
2-mercaptoethanol for PT-18 cells; RPMI-1640 medium supplemented with 0.05 mmol/L 2-mercaptoethanol and 10% FBS for 10P-2,
10P-12, 11PO-1, and CFTL-12 cells; DMEM supplemented with 10% FBS for
P815 cells; and Eagle's minimum essential medium supplemented with
Earle's balanced salt solution, 16% FBS, 0.1 mmol/L nonessential amino acids, and 2 mmol/L L-glutamine for RBL-1 and RBL-2H3 cells. All
media contained penicillin and streptomycin at a concentration of 100 IU/mL and 100 µg/mL, respectively.
Preparation of MCs.
Murine mast cell populations, ie, peritoneal mast cells (PMCs) and bone
marrow-derived cultured mast cells (BMCMCs), were prepared as described
previously.21,22 To obtain PMCs, retired breeder Balb/c
mice (The Jackson Laboratory, Bar Harbor, ME) were euthanized by cervical dislocation, and 10 mL sterilized lavage solution was injected into the abdominal cavity. After 3 minutes of
abdominal massage, the abdominal cavity was opened and the fluid was
collected. Total peritoneal cells from intraperitoneal lavage solution
were fractionated on 23% (wt/vol) metrizamide (Sigma). The MC content
of mast cell enriched fractions was determined by Toluidine-blue
staining (>95% purity)23; viability of all fractions was
determined by Trypan-blue dye exclusion (>95% viability).
IL-3-dependent BMCMCs were derived from the femoral bone marrow cells
of Balb/c mice and were maintained in DMEM supplemented with 10% FBS,
2 mmol/L L-glutamine, 0.05 mmol/L 2-mercaptoethanol, and 20% (vol/vol)
supernatants of concanavalin-A-activated spleen cells.22
Measurement of 45Ca uptake by MCs.
Cells were plated into MultiScreen-DV 96-well filtration plates
(Millipore, Marlborough, MA) at a density of 5 to 10 × 104 cells/well in 100 µL serum-free DMEM and were then
incubated in a total volume of 0.25 mL of serum-free DMEM (containing
1.8 mmol/L CaCl2) in the presence of 0.25 mg/mL bovine
serum albumin (BSA; Sigma; included to stabilize the compounds in the
aqueous solution), 1 µCi/mL 45Ca, and increasing
concentrations of the different compounds for 30 minutes at
37°C.24,25 This incubation period was chosen, because
in a control experiment, in which the time course of the capsaicin
induced 45Ca uptake was determined, no major differences
were found between the values measured on DRGs and mast cells (the
45Ca uptake reached its maximum value after 12 and 16 minutes, respectively). Cells were then washed five times with ice-cold
serum-free DMEM by filtration using a MultiScreen Vacuum Manifold
(Millipore). Filters were dried under a heat lamp and punched out into
scintillation vials using MultiScreen disposable punch tips, and the
radioactivity was determined by scintillation counting. For each data
point in each experiment, five wells were assayed. In the case of
desensitization experiments, cells were incubated at 37°C with
capsaicin or RTX for the indicated period of time (usually 6 hours) and
then challenged with capsaicin or RTX, as indicated.
Analysis of 45Ca uptake data.
The 45Ca uptake experiments were analyzed as described
previously25 by computer fit to the Hill
equation.26 In the case of the desensitization experiments,
desensitization was defined as the difference (in disintegrations per
minute [dpm] per well) between the increase in
45Ca uptake in these and in control cells upon challenge by
capsaicin or RTX. The decrease in the 45Ca uptake induced
by vanilloids was plotted against the pretreatment concentration of RTX
or capsaicin and the data were fitted to the Hill equation. Data from
competition experiments, in which the effect of the desensitizing
compound was antagonized by either a competitive (capsazepine) or a
noncompetitive (ruthenium red) antagonist, were fitted to the modified
Hill equation.27 Data fitting was performed using the
computer program MicroCal Origin 3.5 (MicroCal Software Inc,
Northampton, MA).
Cytotoxicity (MTT) assay.
Cytotoxicity was measured with the MTT assay kit according to the
manufacturer's protocol (Sigma). Briefly, cells were incubated for 6 hours at 37°C with the compound as indicated. Cells were then
washed with RPMI-1640 medium without phenol red (GIBCO BRL), resuspended in the same medium at a final concentration of 1 × 105 cells/mL, and plated into 96-well plates. Tetrazolium
salt (MTT) was added to each well at the concentration of 0.5 mg/mL,
and the absorbance of the developed purple color was measured at 567 nm.
Release of serotonin by MCs.
Degranulation of MCs was assessed by
[3H]5-hydroxytryptamine ([3H]-5HT;
Dupont-NEN) release as described previously.28 Briefly, cells in 48-well plates (105 cells/well) were preincubated
with [3H]-5HT (1 µCi/mL) in Tyrode's buffer for 2 hours at 37°C and then were washed three times with buffer to
remove excess radioactivity. The cells were then challenged for 30 minutes at 37°C with different agents and supernatants were
collected by centrifugation. Radioactivity released into the
supernatants was measured by scintillation counting.
Determination of cytokine secretion.
The production of IL-4, IL-6, and TNF- was determined by
enzyme-linked immunosorbent assay (ELISA) kits (Biosource
International, Camarillo, CA). Cells were plated into 48-well plates in
serum-free DMEM and incubated with vanilloids for different times
(usually 3 hours) at 37°C, after which the supernatant was
collected in a final volume of 0.5 mL. ELISAs were performed according
to the manufacturer's protocols.
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RESULTS |
Capsaicin and resiniferatoxin induce 45Ca uptake in MC
lines.
A variety of MC lines were tested for stimulation of 45Ca
uptake in response to 3 µmol/L capsaicin (a concentration that
induces a maximal response in DRGs10,25). As shown in
Fig 1, 6 of 9 mast cell lines examined
exhibited significant stimulation of 45Ca uptake after the
addition of capsaicin. Adenosine-5-triphosphate (ATP; 500 µmol/L),
which has been shown to activate a nonspecific, Ca2+-permeable channel found on many cell types, including
mast cells,29 was used as a positive control. ATP induced
45Ca uptake in each of the cell lines we examined,
including the RBL-1, RBL-2H3, and CFTL-12 cell lines, which showed no
sensitivity to capsaicin.
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| Fig 1.
Capsaicin induces 45Ca uptake in different MC
lines. Cells were challenged with 3 µmol/L capsaicin for 30 minutes
to induce 45Ca uptake. Points represent mean over baseline
values from sets of five determinations in at least three experiments
in each case; error bars indicate SEM.
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To compare the pharmacologic characteristics of the vanilloid
responsiveness of the MC lines with those of DRGs, we determined the
dose-response curves for capsaicin- and RTX-induced 45Ca
uptake. In the MC/9 cells, capsaicin induced 45Ca uptake in
a dose-dependent fashion (Fig 2), with a
Kd value of 0.661 ± 0.04 µmol/L (mean ± SEM for 4 experiments). The stimulation of 45Ca uptake by capsaicin
was noncooperative, with a Hill coefficient of 0.97 ± 0.01 (mean ± SEM for 4 experiments). Similar Kd and Hill
coefficient values were found on the other responsive cell lines; the
respective average values for 10P-2 cells were 0.552 µmol/L and
0.967, for 10P-12 cells were 0.494 µmol/L and 1.06, for 11PO-1 cells
were 0.683 µmol/L and 0.993, for P815 cells were 0.590 µmol/L and
1.01, and for PT-18 cells were 0.393 µmol/L and 1.03 (2 to 3 experiments for each cell line). These values agreed well with those
determined previously on rat DRGs10
(Table 2).
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| Fig 2.
Comparison of dose-response curves for induction of
45Ca uptake by capsaicin or RTX in MC/9 cells. Cells were
challenged with different concentrations of capsaicin ( ) or RTX
( ) for 30 minutes. Points represent mean values from sets of five
determinations in a single experiment; error bars indicate SEM. In both
cases, at least three experiments yielded similar results. The
theoretical curves were calculated by fitting the measured values to
the Hill equation.
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RTX induced 45Ca uptake into mast cells with markedly
higher potency than did capsaicin (Kd = 2.1 ± 0.18 nmol/L, mean ± SEM for 4 experiments on MC/9 cells; Fig 2). As was
the case with capsaicin, the stimulation of 45Ca uptake by
RTX was noncooperative (Hill coefficient, 0.82 ± 0.06; mean ± SEM for 4 experiments on MC/9 cells). Once again, these values agreed
well with those determined on DRGs (Table 2 and Acs et
al10).
Vanilloid-induced 45Ca uptake in MCs is inhibited by
vanilloid antagonists.
In DRGs, 45Ca uptake in response to vanilloids can be
blocked by the competitive antagonist capsazepine30,31 and
the noncompetitive antagonist ruthenium red.32 In MC/9
cells (and also in the other responding cell lines; data not shown)
capsazepine likewise inhibited the 45Ca uptake by 3 µmol/L capsaicin (Fig 3). The
Ki for inhibition by capsazepine was 0.311 ± 0.03 µmol/L (mean ± SEM for 4 experiments) and reflected
noncooperative kinetics (Hill coefficient, 0.91 ± 0.06; mean ± SEM for 4 experiments). The noncompetitive inhibitor ruthenium red also
inhibited the 45Ca uptake induced by capsaicin (Fig 3); the
ED50 was 0.98 ± 0.07 µmol/L (mean ± SEM for 3 experiments). Similar values were obtained for the inhibition of
45Ca uptake induced by 4 nmol/L RTX by capsazepine and
ruthenium red (Ki of 0.291 ± 0.05 µmol/L
and ED50 of 0.91 ± 0.11 µmol/L for capsazepine and
ruthenium red, respectively; mean ± SEM for 3 experiments in each
case). These values once again showed good agreement with those found
for rat DRGs (Table 2 and Acs et al10).
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| Fig 3.
Vanilloid antagonists inhibit the capsaicin-induced
45Ca uptake in MC/9 cells. Cells were challenged with 3 µmol/L capsaicin in the presence of increasing concentrations of
capsazepine () or ruthenium red (). Points represent mean values
from sets of five determinations in a single experiment; error bars
indicate SEM. Two additional experiments in each case yielded similar
results. The theoretical curves were calculated by fitting the measured values to the Hill equation.
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Pretreatment of MCs with capsaicin or RTX results in desensitization.
In DRGs, stimulation of 45Ca uptake by capsaicin is
followed by desensitization of subsequent stimuli.2,10 We
therefore examined the effect of vanilloid pretreatment on
vanilloid-induced activation of MCs. MC/9 cells were pretreated with
different concentrations of capsaicin for 6 hours (a time that was
shown to induce maximal desensitization on rat DRGs10) and
challenged with 3 µmol/L capsaicin to evoke 45Ca uptake.
Desensitization was determined as a decrease in the capsaicin induced
response compared with control (solvent-treated) cells. Pretreatment of
the cells with capsaicin resulted in the dose-dependent loss of
45Ca uptake induced by challenge with 3 µmol/L capsaicin
(Fig 4). The ED50 and Hill
coefficients were 0.525 ± 0.01 µmol/L and 1.06 ± 0.05, respectively (mean ± SEM for 3 experiments). These values are similar to those for induction of desensitization on DRGs. Likewise, they resemble those for the acute induction of
45Ca uptake by capsaicin10 (Table 2).
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| Fig 4.
Pretreatment with capsaicin or RTX results in the
desensitization of capsaicin-induced 45Ca uptake in MC/9
cells. Cells were pretreated with different concentrations of capsaicin
() or RTX () for 6 hours and then were challenged with 3 µmol/L
capsaicin for 30 minutes. For better comparison, the dose-response for
RTX-induced desensitization on DRGs is also plotted (dotted line).
Desensitization was defined as the difference (in dpm per well) in
45Ca uptake between pretreated and control cells when
challenged with capsaicin. Points represent mean values from sets of
five determinations in a single experiment; error bars indicate SEM. In
both cases, at least three experiments yielded similar results. The
theoretical curves were calculated by fitting the measured values to
the Hill equation. The dose-response curve for RTX-induced desensitization on DRGs is a single experiment, yielding similar values
to those found by us previously.10
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When pretreated for 6 hours with increasing concentrations of RTX, MC/9
cells also exhibited desensitization to subsequent challenge with 3 µmol/L capsaicin (Fig 4). The ED50 and Hill coefficient values for desensitization by RTX were 2.3 ± 0.65 nmol/L and 0.84 ± 0.07, respectively (mean ± SEM for 4 experiments). The
potency of RTX thus was markedly different from that determined on rat DRGs for desensitization (ED50 of 81 ± 5 pmol/L; see
Acs et al10). However, it was similar to that for induction
of 45Ca uptake by RTX on MCs (ED50 of 2.1 ± 0.18 nmol/L; see above). In addition, RTX induced desensitization in a
noncooperative manner, once again similar to the induction of
45Ca uptake by RTX in these cells but in contrast to the
positive cooperativity found on DRGs (Hill coefficient, 1.51 ± 0.11; see Acs et al10).
MC lines express the C-type but not the R-type vanilloid receptor.
Previously, we have shown high-affinity specific binding of
[3H]RTX on DRGs cultured in vitro25 and on
DRG membrane preparations.8,33 Using the same protocols, we
were unable to detect specific binding on any of the mast cell lines
examined, either on intact cells or on membranes. The measured value on
membranes was 0.69 ± 0.99 fmol/mg protein (this value did not
differ significantly from 0; P > .5), in contrast to the 198 ± 13 fmol/mg protein on DRG membranes.25 The lack of
binding is consistent with the lack of the high-affinity binding site.
The failure in detection of the low-affinity, although obviously
existing, C-type receptor with this binding assay was predictable and
could be attributed to the combination of lower affinity, nonspecific
binding, and low Bmax on mast cells (see also in
Discussion).
Table 2 summarizes the characteristics of the vanilloid receptors found
on MC/9 cells and compares them with those described on rat
DRGs.10 Our findings strongly argue that MCs possess the
C-type receptor that can be detected by the 45Ca uptake
assay but not the R-type receptor that can be assayed by
[3H]RTX binding.
Vanilloid-mediated activation of MCs is not associated with
cytotoxicity.
On DRGs, the capsaicin-induced Ca2+ influx is thought to be
a major factor mediating vanilloid cytotoxicity.9,34
Because of the relative resistance of mast cells to vanilloid toxicity in vivo,35,36 we wished to evaluate whether the mast cell
vanilloid receptor confers cytotoxicity in the MCs similar to its
action on DRGs. Using the MTT assay, we found that the exposure of MC/9 cells to capsaicin for 6 hours was nontoxic up to a concentration of 10 µmol/L (data not shown); likewise, RTX showed no toxicity up to 20 nmol/L.
Vanilloids do not induce degranulation in MCs.
To assess whether vanilloid-induced activation of mast cells is
associated with degranulation, we determined the release of [3H]5-HT (serotonin) from MC lines upon vanilloid
treatment. Neither capsaicin nor RTX induced significant
[3H]5-HT release from any of the MC lines examined at
concentrations corresponding to those that induced 45Ca
uptake (data not shown). Up to 10 µmol/L capsaicin (and 20 nmol/L
RTX), the maximal increase was approximately 4% to 5% of the total,
which was not statistically significant (P > .5, Student's t-test) and did not exceed the level of the spontaneous
release. It thus seems that, in the MC lines, the vanilloid receptor
and the mediated influx of calcium are not directly coupled to
degranulation.
Capsaicin induces 45Ca uptake in BMCMCs but not in PMCs.
We have also tested the effect of vanilloids on different mast cell
populations isolated from mice. Capsaicin did not induce 45Ca uptake in PMCs (at concentrations up to 20 µmol/L)
but was effective on BMCMCs (Fig 5). The
effect of capsaicin on BMCMCs was inhibited by the vanilloid
antagonists capsazepine and ruthenium red (10 µmol/L and 5 µmol/L,
respectively), also showing its vanilloid receptor specificity. It is
important to note that the estimated receptor density
(Bmax) on BMCMCs was approximately 10% of the level found
on mast cell lines (thus 1% of that found on DRGs), presumably
reflecting in part the extremely small size of the BMCMCs. On the other
hand, capsaicin was unable to induce degranulation at this (high
nanomolar to low micromolar) concentration range on either
BMCMCs or PMCs, similar to its ineffectiveness on MC lines (see above).
It should be noted that capsaicin did induce significant histamine
release from the cells at concentrations of greater than 50 µmol/L,
and, furthermore, its actions at these high concentrations were not
antagonized by capsazepine (data not shown); it therefore seems
probable that these latter effects reflect nonspecific,
non-vanilloid-mediated actions of the compound.
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| Fig 5.
Capsaicin induces 45Ca uptake in BMCMCs but
not in PMCs. Cells were challenged with different concentrations of
capsaicin (C) for 30 minutes to induce 45Ca uptake. The
vanilloid specificity of the capsaicin-induced response was determined
by using 10 µmol/L capsazepine (CPZ) or with 5 µmol/L ruthenium red
(RR) as vanilloid antagonists. Points represent mean over baseline
values from sets of five determinations in three experiments in each
case; error bars indicate SEM.
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Capsaicin induces the differential production of IL-4.
Because activated MCs are known to produce and secrete a wide array of
proinflammatory cytokines,37 we have assessed the production of TNF-, IL-4, and IL-6 by MCs. As seen in
Fig 6A, capsaicin failed to induce the
release of TNF- from P815 cells, although 100 nmol/L phorbol
12-myristate 13-acetate (PMA) was found to be effective; similarly,
capsaicin was ineffective in producing IL-6 (data not shown). On the
other hand, capsaicin did induce the production of IL-4 from P815 cells
(Fig 6B) at concentrations as low as 0.1 µmol/L. The response was
blocked by capsazepine at concentrations from 0.1 to 10 µmol/L (the
latest is plotted in Fig 6), supporting its vanilloid specificity.
Capsazepine alone was ineffective in inducing IL-4 production.
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| Fig 6.
Capsaicin induces the production of IL-4 but not TNF-
in P815 cells. Cells were treated with different concentrations of capsaicin (C) for 3 hours. Solvent (Base) or 100 nmol/L PMA-treated cells were used as controls. The inhibitory effect of capsazepine (CPZ)
was assessed by incubating one group of cells with 10 µmol/L CPZ in
the presence of 10 µmol/L capsaicin. Supernatants were then collected
and the produced TNF- (A) or IL-4 (B) content was determined in
duplicates by ELISA kits according to the manufacturer's protocol.
Each of the measured doses of capsaicin caused significant release of
IL-4 (*P < .01). CPZ significantly (**P < .05)
decreased the release of IL-4 by capsaicin, whereas it did not modify
the release (***P > .5) when applied alone (Student's
t-test). Points represent mean values of three individual
experiments; error bars indicate SEM.
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| |
DISCUSSION |
We report here for the first time that a variety of murine mast cell
lines as well as BMCMCs express functional vanilloid receptors. We have
also shown that the MC vanilloid receptor is similar to the C-type
vanilloid receptor described on DRGs10 and that its
activation leads to Ca2+ uptake, desensitization, and
differential cytokine production in MCs. Our findings that vanilloids,
in addition to the activation of a subset of sensory neurons, also can
act directly on nonneuronal cell types suggest the need to reexamine
the complex in vivo effects of vanilloids, particularly with respect to
neurogenic inflammation.
In our previous studies,10 we had shown the existence of
two pharmacologically defined classes of vanilloid receptors (the R- and the C-type) on rat DRGs (Table 1). In MCs, the
vanilloid-induced 45Ca uptake showed similar pharmacologic
behavior to that found on DRGs (Table 2), suggesting that (1) certain
MCs possess the C-type vanilloid receptor (a receptor subtype that
functions as a ligand-gated, Ca2+ permeable
channel9) and (2) the characteristics of the C-type receptors found on MC lines are very similar to those on
DRGs.10 On the other hand, we failed to detect any specific
[3H]RTX binding on MCs (either on intact cells or on
membrane preparations) with techniques optimized for
[3H]RTX binding on DRGs. This result, in addition to the
similar affinities of RTX on inducing 45Ca uptake and
desensitization, compared with its orders of magnitude higher potency
to induce desensitization (and binding) than 45Ca uptake on
DRGs10,25 (see also in Tables 1 and 2), suggests that MCs
do not express the R-type vanilloid receptor. The existence of the
C-type and lack of the R-type receptor on mast cells further supports
this subclassification of vanilloid receptors. Our failure to detect
the low-affinity [3H]RTX binding to the C-type receptor
is presumably attributable to several factors, including the relatively
low density of C-type receptors on mast cells (the capsaicin-induced
45Ca uptake in mast cells is approximately 10% of that in
DRGs and even lower [<1%] on BMCMCs) and the predicted 45-fold
weaker binding affinity to the C-site (the ratio of the
ED50s for 45Ca uptake and [3H]RTX
binding on DRGs, 2.1/0.047  45). These two factors suggest a
450-fold less favorable ratio of specific to nonspecific binding for
the C-type receptor on mast cells than for the R-type receptor on
DRGs.25 Finally, the [3H]RTX binding assay is
significantly improved by the use of 1-acid glycoprotein
to remove nonspecifically bound [3H]RTX, relying on the
slower off rate of the specifically bound [3H]RTX than of
the nonspecifically bound ligand.38 A faster off-rate of
the ligand from the C-type receptor would prevent the use of 1-acid glycoprotein, further degrading the detection of
specifically bound [3H]RTX.
In DRGs, the prolonged action of capsaicin results in the degeneration
of neurons due to calcium-mediated cell toxicity.9,34 However, 6 hours of treatment of MCs with capsaicin up to 10 µmol/L showed no toxicity (Fig 5). This result is in good accord with the in
vivo finding that neonatal capsaicin treatment, which causes the
degeneration and ablation of sensory neurons,4 does not decrease the number of mast cells in the gastric mucosa,19
dura mater,36 lung, or spleen.35 The lack of
toxicity after the activation of the capsaicin-sensitive channel and
the concomitant increase of intracellular Ca2+
concentration is probably due to the different Ca2+
handling mechanisms in mast cells.
Interestingly, capsaicin was effective in inducing 45Ca
uptake in most, but not all, of the MC lines examined. Although the reason for the latter observation remains to be determined, our findings clearly show that distinct murine MC populations express the
C-type vanilloid receptor. We also found that BMCMCs, but not PMCs of
Balb/c mice, exhibited 45Ca-uptake after stimulation with
capsaicin. These findings may indicate that the expression of the
vanilloid receptor is developmentally and/or
microenvironmentally regulated. BMCMCs (considered to be similar to
mucosal-type MCs) differ from connective-tissue type MCs isolated from
the serosal peritoneum of mice in numerous characteristics, including
mediator content, responses to pharmacologic agents, and sensitivity to
agents that induce proliferation, activation, or mediator
release.39 For example, IL-3 has been shown to induce proliferation of BMCMCs but not PMCs isolated from Balb/C
mice,40 whereas stem cell factor (SCF) reportedly induces
degranulation of murine PMCs but not BMCMCs.41,42
Accordingly, it will be of interest to assess the expression of
vanilloid receptors and the response to vanilloids in various
distinct MC populations of various maturation states.
To address the biologic significance of capsaicin-induced
45Ca uptake in MCs, we next assessed whether activation of
the MC vanilloid receptor would induce the release of preformed,
granule-associated MC mediators or the production of proinflammatory
cytokines. To our surprise, neither capsaicin nor RTX caused vanilloid
receptor-mediated degranulation in any of the responsive MC lines or
MCs. Only when stimulated with high micromolar to
millimolar concentrations of capsaicin did MCs exhibit
significant release of serotonin, which, taken together with the
ineffectiveness of capsazepine in reducing this response, suggests an
unspecific, nonvanilloid receptor-mediated mechanism of MC activation
at these high vanilloid concentrations.
In contrast to its lack of effect on degranulation, capsaicin induced
production of IL-4 in the P815 clone. This response was dose-dependent
and observed at the same concentrations of capsaicin as
45Ca uptake. In addition, coincubation with antagonists
capsazepine or ruthenium red significantly inhibited IL-4 production,
further supporting a vanilloid receptor-mediated mechanism of MC
activation. Given the ability of MC-derived cytokines to influence
diverse biologic responses,43 the identification of agents
that induce MC cytokine production in the absence of (potentially
counterproductive) degranulation is of particular interest. To our
knowledge, only lipopolysaccharide, prostaglandin E2,
cholera toxin, and SCF have been shown to induce selective stimulation
of MC cytokine production independently of histamine
release.42 Moreover, MCs produced IL-4 upon challenge with
capsaicin that resulted in little or no detectable release of TNF-
or IL-6. This observation confirms previous reports suggesting
different regulatory mechanisms in MCs for the expression of different
groups of cytokines. Stimulation of BMCMCs with protein kinase C
activators such as PMA reportedly induces the expression of TNF- and
IL-6 but not of IL-2, IL-3, IL-4, IL-5, granulocyte-macrophage
colony-stimulating factor (GM-CSF), or interferon- (IFN-), and SP
has been shown to differentially stimulate the secretion of TNF- but
not that of IL-1, IL-3, IL-4, IL-6, or GM-CSF in freshly isolated
murine PMCs.13,21,44
Although the in vivo relevance of our findings remains to be
elucidated, the observation that capsaicin in vitro can induce MCs to
produce significant amounts of IL-4, but not TNF- or IL-6, in the
absence of degranulation (but with electrophysiological alterations,
ie, 45Ca uptake) and, furthermore, that capsaicin
selectively affects different MC populations (ie, BMCMCs v
PMCs) offers a new look at the potential physiologic functions of MCs.
Vanilloid receptor signaling of MCs may play a role in MC-directed
regulation of antibody production and inflammation and the development
of effector T-cell responses, as well as in the autocrine modification
of MC functions.45,46 Moreover, the lack of in vitro
toxicity of vanilloids on MCs versus DRGs and the differential in vivo action of capsaicin on MCs (namely neonatal capsaicin treatment affects
exclusively MCs expressing markers of MMCs35,36) suggests the possible involvement of vanilloid receptors in the development and
differentiation of MCs.
The molecular cloning of the vanilloid receptor would have great impact
on the detailed evaluation of vanilloid-mediated mechanisms. A major
obstacle has been the lack of a convenient source of RNA and DNA. The
establishment of neuronal hybrid cell lines derived from
DRGs47,48 has not proved to be a good solution, because these cell lines tend to lose their vanilloid sensitivity. Thus, our
description of cell lines expressing the C-type vanilloid receptors
should greatly facilitate aspects of the molecular characterization of
these receptors.
The existence of vanilloid receptors on MCs (and other nonneuronal cell
types, characterization of which is currently in progress; T. Bíró, manuscript in preparation)
necessitates the reevaluation of our understanding of the in vivo
pathways of vanilloid action (Fig 7).
Previously, capsaicin and related compounds were believed to activate
exclusively neurons expressing the vanilloid receptors, and the
modification of the functions of nonneural cell types, including MCs,
was regarded as an indirect process via the release of peptide
transmitters.2,18,19 Our demonstration that capsaicin can
act directly on MCs in parallel with the activation of sensory neurons
shows a potential new aspect of the processes induced by vanilloids,
eg, neurogenic inflammation7 and stimulation of murine hair
growth in vivo.49 In addition, the identification of
vanilloid receptors on easily cultured cells should be a valuable experimental tool in the effort to identify the putative endogenous ligand(s) for the vanilloid receptor.
View larger version (80K):
[in this window]
[in a new window]
| Fig 7.
A new model for the action of capsaicin and related
vanilloids on MCs. Capsaicin, in addition to its sensory
neuron-mediated effect via C- and R-type vanilloid receptors (C-r and
R-r), can also directly act on mast cells expressing functional
(C-type) vanilloid receptors.
|
|
These findings might have therapeutic implications. Capsaicin is widely
used in therapy in various inflammatory conditions, including
rheumatoid arthritis, psoriasis, neuropathy of different origins,
prurigo, urticaria, and atopic eczema, in which an increased number of
mast cells and/or their altered, pathognomic function have been
described.50,51 The efficacy of capsaicin in these conditions might be augmented by the accompanied modification of the
function of MCs. In addition, the presence of a new receptor on MCs may
further help in understanding the role of MCs as a central component of
the neuroimmune axis52 and to clarify the functional
significance of vanilloid receptor-dependent signaling in health and
disease.
| |
FOOTNOTES |
Submitted August 4, 1997;
accepted October 2, 1997.
Supported in part by a grant from the Deutsche Forschungsgemeinschaft
(DFG Pa 345/6-1) to R.P.
Address reprint requests to Peter M. Blumberg, PhD, MMTP/LCCTP/NCI,
Bldg 37, Room 3A01, 37 Convent Dr MSC 4255, Bethesda, MD 20892-4255.
The publication costs of this article were defrayed in part by page
charge payment. This 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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S.-Y. Choi, H. Ha, and K.-T. Kim
Capsaicin Inhibits Platelet-Activating Factor-Induced Cytosolic Ca2+ Rise and Superoxide Production
J. Immunol.,
October 1, 2000;
165(7):
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[Abstract]
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M. Oortgiesen, B. Veronesi, G. Eichenbaum, P. F. Kiser, and S. A. Simon
Residual oil fly ash and charged polymers activate epithelial cells and nociceptive sensory neurons
Am J Physiol Lung Cell Mol Physiol,
April 1, 2000;
278(4):
L683 - L695.
[Abstract]
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M. A. Schumacher, I. Moff, S. P. Sudanagunta, and J. D. Levine
Molecular Cloning of an N-terminal Splice Variant of the Capsaicin Receptor. LOSS OF N-TERMINAL DOMAIN SUGGESTS FUNCTIONAL DIVERGENCE AMONG CAPSAICIN RECEPTOR SUBTYPES
J. Biol. Chem.,
January 28, 2000;
275(4):
2756 - 2762.
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S.-Y. Choi and K.-T. Kim
Capsaicin Inhibits Phospholipase C-Mediated Ca2+ Increase by Blocking Thapsigargin-Sensitive Store-Operated Ca2+ Entry in PC12 Cells
J. Pharmacol. Exp. Ther.,
October 1, 1999;
291(1):
107 - 114.
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T. Cao, N. P. Gerard, and S. D. Brain
Use of NK1 knockout mice to analyze substance P-induced edema formation
Am J Physiol Regulatory Integrative Comp Physiol,
August 1, 1999;
277(2):
R476 - R481.
[Abstract]
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A. Szallasi and P. M. Blumberg
Vanilloid (Capsaicin) Receptors and Mechanisms
Pharmacol. Rev.,
June 1, 1999;
51(2):
159 - 212.
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Z. Olah, T. Szabo, L. Karai, C. Hough, R. D. Fields, R. M. Caudle, P. M. Blumberg, and M. J. Iadarola
Ligand-induced Dynamic Membrane Changes and Cell Deletion Conferred by Vanilloid Receptor 1
J. Biol. Chem.,
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Abstract
Introduction
Abstract