Direct Neurite-Mast Cell Communication In
Vitro Occurs Via the Neuropeptide Substance
P
This information is current as
of June 18, 2017.
R. Suzuki, T. Furuno, D. M. McKay, D. Wolvers, R.
Teshima, M. Nakanishi and J. Bienenstock
J Immunol 1999; 163:2410-2415; ;
http://www.jimmunol.org/content/163/5/2410
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References
Direct Neurite-Mast Cell Communication In Vitro Occurs Via
the Neuropeptide Substance P1
R. Suzuki,* T. Furuno,* D. M. McKay,‡ D. Wolvers,‡ R. Teshima,† M. Nakanishi,2* and
J. Bienenstock3†
D
uring the last decade, there has been an exponential increase in data illustrating that the immune and nervous
systems are not disparate entities (1, 2). The mast cellnerve relationship has served as a prototypic association and has
provided substantial evidence for bidirectional communication between nerves and immune cells (3). Early studies elegantly described the nonrandom spatial association of mast cells and nerves
in a variety of tissues in which actual membrane-membrane contacts could be observed (4, 5). Many studies have shown that messenger molecules (e.g., neuropeptides) from nerves can elicit mast
cell degranulation (6) and that mast cell-derived mediators (e.g.,
histamine, serotonin, and platelet-activating factor) modulate neurotransmission (7, 8). For instance, intestine from b-lactoglobulin
(b-LG)-sensitized animals respond to in vitro b-LG challenge with
an increase in electrogenic chloride secretion that is largely mast
cell-mediated (9). The same study showed that the chloride secretory event (as evidence of mast cell activation) was precisely correlated with a concomitant release of acetylcholine and that the
release of the neurotransmitter was mimicked by the application of
exogenous histamine. Perdue et al. (10) reported that the transient
*Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan; †National Institute of Health Sciences, Tokyo, Japan; and ‡Intestinal Disease Research
Program, McMaster University, Hamilton, Canada
increase in ion transport evoked by electrical field stimulation of
nerves in jejunal segments from mast cell-deficient (W/Wv) mice
was half that which occurred in congenic mast-cell containing
mice. Furthermore, reconstitution of the W/Wv mice with mast
cells resulted in a restoration of a normal ion transport response to
nerve stimulation. Also, Theoharides and colleagues (11) found
that electrical nerve stimulation caused mast cell degranulation in
the dura mater.
However, while these tissue studies have shown a functional
nerve-mast cell interplay, they do not dismiss the possibility that
an intermediate cell transduces or modulates the nerve-mast cell
communication. It is this question that is the crux of the present
study. Using an in vitro coculture model comprising rat basophilic
leukemic cells (RBLs),4 which display properties of mucosal-type
mast cells, and neurite-sprouting murine superior cervical ganglia
(12), we have examined direct nerve-mast cell communication.
Our findings demonstrate that mast cell activation, as judged by
calcium mobilization, can be a direct consequence of contact with
a specific activated nerve fiber. Moreover, we provide evidence
that this RBL cell activation was mediated, at least in part, by the
neuropeptide substance P acting through neurokinin (NK)-1 tachykinin receptors.
Materials and Methods
Received for publication January 6, 1999. Accepted for publication June 10, 1999.
Nerve-RBL cell coculture
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
Following a published protocol (12, 13), superior cervical ganglia (SCG)
were dissected from newborn (0 – 48 h old) CBA mice (Japan SLC, Shizuoka, Japan) and rinsed in HBSS containing 10 mM HEPES (pH 7.4).
Each ganglion was divided into two to four pieces and incubated for 60 min
at 37°C in 2 ml of HEPES containing 0.125% trypsin (grade II; Sigma, St.
Louis, MO). The resultant cell suspension was plated at a density of 0.5–
1 3 104 nerve cells onto matrigel (Becton Dickinson, Bedford, MA)-coated 35-mm diameter glass dishes. The neurons were grown in F12 culture
1
This work was supported by research funds from the Ministry of Education of Japan
(to M.N.) and Medical Research Council (Canada) operating grants (to J.B. and
D.M.M.).
2
Address correspondence and reprint requests to Dr. M. Nakanishi, Department of
Analytical Chemistry and Biophysics, Faculty of Pharmaceutical Sciences, Nagoya
City University, Tanabe-dori, Mizuho-ku, Nagoya 467, Japan. E-mail address:
[email protected]
3
Address correspondence and reprint requests to Dr. J. Bienenstock, Intestinal Disease Research Program, McMaster University, HSC-3N21, 1200 Main Street West,
Hamilton, Ontario, Canada L8N 3Z5. E-mail address: [email protected]
Copyright © 1999 by The American Association of Immunologists
4
Abbreviations used in this paper: RBL, rat basophilic leukemic cell; BK, bradykinin;
NK, neurokinin; SCG, superior cervical ganglia; SV, scorpion venom; NGF, nerve
growth factor.
0022-1767/99/$02.00
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Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. However, whether mast
cell activation occurs as a direct response to neuronal activation or requires an intermediary cell is unclear. Addressing this issue,
we used an in vitro coculture approach comprising cultured murine superior cervical ganglia and rat leukemia basophilic cells
(RBLs; possesses properties of mucosal-type mast cells). Following loading with the calcium fluorophore, Fluo-3, neurite-RBL
units (separated by <50 nm) were examined by confocal laser scanning microscopy. Addition of bradykinin, or scorpion venom,
dose-dependently elicited neurite activation (i.e., Ca21 mobilization) and, after a lag period, RBL Ca21 mobilization. Neither
bradykinin nor scorpion venom had any direct effect on the RBLs in the absence of neurites. Addition of a neutralizing substance
P Ab or a neurokinin (NK)-1 receptor antagonist, but not an NK-2 receptor antagonist, dose-dependently prevented the RBL
activation that resulted as a consequence of neural activation by either bradykinin or scorpion venom. These data illustrate that
nerve-mast cell cross-talk can occur in the absence of an intermediary transducing cell and that the neuropeptide substance P,
operating via NK-1 receptors, is an important mediator of this communication. Our findings have implications for the neuroimmune signaling cascades that are likely to occur during airways inflammation, intestinal hypersensitivity, and other conditions in
which mast cells feature. The Journal of Immunology, 1999, 163: 2410 –2415.
The Journal of Immunology
medium (Life Technologies, Rockville, MD) supplemented with 0.2 mM
L-glutamine, 0.3% glucose, 3% antibiotic/antimycotic (A-7292) (all from
Sigma), 10% FBS (BioWhittaker, Walkersville, MD), and 50 ng/ml murine
nerve growth factor (NGF, 2.5S; Upstate Biotechnology, Lake Placid, NY).
Nonganglionic cells were killed by an initial exposure to cytosine-b-Darabinofuranoside (Ara-C, 1026M; Sigma) for 24 h.
RBLs (RBL-2H3, these cells display some of the properties of mucosal
mast cells and are generally accepted as a model of mucosal-type mast
cells) were originally a gift from Dr. R. P. Siraganian (National Institutes
of Health, Bethesda, MD) and were maintained in F12 culture media containing 50 ng/ml NGF (RBLs cultured in NGF-free medium displayed
similar cell growth and morphology to those maintained in NGF-containing medium). The pattern of b-hexosaminidase secretion from the RBLs
used in this study was similar to previous studies with this cell clone (14).
For coculture experiments, RBLs (104/dish) were added to 48-h-old cultures of SCG-neurites and incubated at 37°C for 72 h.
Cellular activation
Mechanism of neurite-to-RBL communication
The neuropeptide substance P was examined as the possible neurite-derived mediator responsible for the RBL activation.
Immunohistochemistry. Seventy-two-hour neurite cultures were fixed for
10 min in 0.05% gluteraldehyde, rinsed, and then preincubated in 5% (w/v)
BSA for 30 min. Cells were then incubated for 24 h at 4°C with primary
rabbit anti-substance P Ab (1 mg/ml diluted in PBS/1% BSA/5% normal
mouse serum). Substance P immunoreactivity was detected by incubation
with goat-anti-rabbit Ab-HRP conjugates for 45 min at room temperature
and visualized with diaminobenzidine. The degree of positivity was assessed by a single investigator in a blinded fashion. All of the appropriate
controls were performed, including omission of the primary antisera, substitution of rabbit non-specific IgG (Dako, Carpinteria, CA) for the primary
Ab, and inclusion of mouse gut tissue as a positive control for substance P
immunostaining.
Inhibitor studies. An affinity column-purified anti-substance P rabbit IgG
polyclonal Ab at 0.2–20 mg/ml was added to neurite-RBL cocultures 20
min before BK or SV stimulation, and cellular responses were measured
microscopically. This Ab has no cross-reactivity with neurokinin A, or
other tachykinins, and has been used as a neutralizing Ab in in vivo studies
(A. Stanisz, unpublished observation). Controls included the use of an irrelevant rabbit IgG (20 mg/ml) and addition of the anti-substance P Ab to
single cultures of neurites or RBLs only. A similar approach was used to
investigate the tachykinin receptor subtype involved in this communication. Before neurite stimulation, cocultures were pretreated with the nonpeptide NK-1 receptor antagonist, CP-99,994-1 (1–100 ng/ml; Pfizer, Groton, CT) (this compound is an upgrade on the NK-1 antagonist, CP-96,345,
which has been shown to block the effects of substance P (19, 20)) or an
NK-2 receptor antagonist, SR 48968 (1 mg/ml; Sonofi Roche, Basel, Switzerland). Both agents were gifts to Dr. A. Stanisz (McMaster University,
Hamilton, Canada), and the concentrations used were based on unpublished in vivo and in vitro studies in Dr. Stanisz’s laboratory. Both NK-
receptor antagonists were tested in single neurite and RBL cultures stimulated with BK or anti-IgE receptor Abs, respectively.
Data presentation
Data are presented as arbitrary fluorescence units or as the percentage of
RBLs responding to simulation (this is an all or nothing measure, based on
Fluo-3 activity), where n values represent the number of cells or the number of RBL-neurite cultures examined.
Results
Effect of nerve stimulation on RBL Ca21 mobilization
Bradykinin. Addition of BK to SCG cultures resulted in a dosedependent neurite activation: 1 nM BK 5 25.6 6 5.8, 10 nM
BK 5 34.2 6 4.9, and 100 nM BK 5 38.9 6 7.6 units of fluorescence intensity/neurite (n 5 7; Fig. 1, A and B), but did not
evoke an increase in fluorescence when added directly to Fluo-3loaded RBLs in the absence of SCG neurites (n . 100 RBLs
examined). Addition of BK (10 nM) to SCG cultures only, resulted
in an increase in neurite fluorescence within 5 s that peaked within
15 s. In coculture studies, BK-induced neurite activation was invariably followed by RBL activation, as indicated by increased
fluorescence (Fig. 1B). The number of RBLs responding increased
in accordance with increasing concentrations of BK (Fig. 1C). The
interval between neurite activation and RBL activation was 5.0 6
1.0, 4.4 6 2.5, and 3.8 6 1.2 s for 1, 10, and 100 nM BK, respectively (n 5 9–20). Additionally, RBLs in contact with BK (10
nM)-stimulated neurites displayed cell membrane ruffling, and cell
size was increased by 19.4 6 2.7% (n 5 7). In the absence of
neurites, BK had no direct effect on RBL membrane morphology
or size. In addition, RBLs (n . 50) retrieved after 72 h of coculture
and examined in isolation were, like naive RBLs, unresponsive to
direct BK (10 or 100 nM) stimulation, as gauged by Ca21 signaling (data not shown).
Scorpion venom. SV also caused an almost instantaneous and
dose-dependent increase in neurite fluorescence, which was maximal within 5 s of addition of the venom (Fig. 2). Thus, 100 pg/ml
or 10 pg/ml SV caused an average increase in neurite fluorescence
of 30.0 6 5.5 and 25.9 6 3.5 units of fluorescence, respectively
(n 5 7); at 1 pg/ml, SV had negligible effects on neurite activity.
SV (100 pg/ml) added directly to Fluo-3-loaded RBLs only did not
evoke any Ca21 response (n . 100 RBLs examined). When SV
was added to SCG-RBL cocultures, the expected increase in neurite activation (i.e., fluorescence) was followed by an increase in
RBL fluorescence (Fig. 2A). The increase in RBL fluorescence
remained elevated for at least 60 s (end of experiment) after addition of SV to the coculture. In all cocultures examined, SVevoked neurite activation preceded RBL activation, with a dosedependent average lag phase of 8.3 6 5.3 and 3.5 6 1.9 s at
concentrations of 10 and 100 pg/ml, respectively (n 5 8). Concentrations of 10 and 100 pg/ml SV resulted in activation in 67 and
73% of the RBLs, respectively (n 5 15; Fig. 2B). Similar to the
BK experiments, RBLs (n . 50 cells) isolated after 72 h of coculture did not respond directly to 10 or 100 pg of SV (data not
shown).
Mechanism of neurite-RBL communication
Using standard indirect immunohistochemical techniques, 72-h
neurite cultures were consistently positive for substance P-like immunoreactivity (data not shown). Since substance P can cause mast
cell degranulation and at pM doses can prime RBLs for subsequent
activation at a lower threshold (6, 13), we proceeded to assess
substance P as a putative mediator responsible for the direct neurite-RBL communication demonstrated here. Experiments in
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Calcium mobilization and activation of fluorophores were used as an index
of cellular activation and were assessed using confocal fluorescence microscopy (15, 16). After 72 h of coculture, cells were treated with culture
medium containing Fluo-3-AM (2.5 mM for 30 min at 37°C; Molecular
Probes, Eugene, OR) and then washed three times with HEPES buffer.
Cells were observed with a confocal laser scanning microscope (Zeiss,
Oberkochen, Germany; LSM-410; argon ion laser at 488 nm), and images
were captured and analyzed using IBM compatible computer software. In
coculture studies, neurites and RBLs in contact with neurites (defined as
cell membranes within ,50 nm of each other (17)) were examined. In
some studies, RBL size was measured (cell circumference determined by
computer image analysis) and membrane morphology examined as other
indices of RBL activity.
Neurite activation was evoked by either bradykinin (BK; 1–100 nM;
Bachem, Bubendorf, Switzerland) or SV (SV; Leiurus quinquestriatus
herbaeus, 1–100 pg/ml; Sigma). Examining RBL-to-neurite communication, the RBLs were specifically stimulated by adding anti-IgE receptor
Abs (BC4, an ascites solution used at a dilution of 1/100,000; a gift from
Dr. R. P. Siraganian) to the cocultures. This Ab causes histamine release
from RBLs (18).
In additional studies, following 72 h of coculture, RBLs were retrieved,
loaded with Fluo-3, and then treated with BK (10 or 100 nM), SV (10 or
100 pg/ml), or substance P (1026 or 1028 M). Calcium mobilization in
RBLs derived from coculture studies in response to these agents was compared with naive RBLs (i.e., those not cocultured with neurites).
2411
2412
NERVE-MAST CELL INTERACTIONS
FIGURE 1. A, A representative fluorescence image showing the effect
of BK (10 nM) at 10 s after its addition to the SCG-RBL coculture (bar 5
20 mm). B, The sequential (neurite first, then RBL) increase in fluorescence
evoked by BK in SCG-RBL cocultures. C, The percentage of RBLs responding to BK stimulation (n 5 9, 20, and 20 for 1, 10, and 100 nM BK).
which SCG-RBL cocultures were stimulated with BK in the presence of neutralizing anti-substance P Abs revealed that the inclusion of the Abs dose-dependently prevented the RBL activation
(Fig. 3, A and B). Treatment with a control isotype-matched irrelevant Ab (n 5 7) did not affect the RBL changes evoked in response to neurite activation by BK (Fig. 3C). As an additional
control, we tested the ability of the anti-substance P Abs to abro-
gate the increase in RBL fluorescence evoked by anti-IgE receptor
Abs. Inclusion of the anti-substance P Abs (20 mg/ml) (or an NK-1
receptor antagonist) did not affect the increase in RBL fluorescence
elicited directly by anti-IgE receptor Abs: 127.0 6 7.1, 124 6 7.2,
122.3 6 7.2 fluorescence units for anti-IgE receptor Ab-treated
RBLs, and time-matched cultures, including an anti-substance P
Ab or the NK-1 receptor antagonist, respectively (n 5 10). These
data confirm the postulate that the effect of the anti-substance P
Abs in the coculture model was due to their substance P-neutralizing effects and not a nonspecific effect on the RBL. Also, the
anti-substance P Ab had negligible effects on BK-induced neurite
activation (data not shown).
Next, we examined the ability of tachykinin receptor antagonists
to abrogate the neurite-RBL communication. Inclusion of the
NK-1 receptor antagonist, CP99,994-01, in the SCG-RBL cocultures did not affect the neurite activation evoked by BK, but significantly and dose-dependently inhibited the subsequent increase
in RBL activation (Fig. 4, A and B). In contrast, the NK-2 receptor
antagonist, SR48,968 (1 mg/ml), did not affect either BK-stimulated neurite activity or the subsequent Ca21 mobilization in RBLs
in coculture studies (Fig. 4C). The receptor antagonists did not
directly affect neurite stimulation or the RBLs when incubated in
the absence of coculture. Also, coculture with neurites did not
enhance the RBL responsiveness to exogenous substance P when
the RBLs were examined in isolation after coculture. Thus, 51 and
21% of RBLs retrieved from cocultures responded with an increase in Ca21 mobilization in response to 1026 M and 1028 M
substance P, respectively, as compared with 50 and 18% of naive
RBLs. The response of both naive and cocultured RBLs to exogenous substance P was blocked (i.e., ,5% of cells responded) by
a 20-min pretreatment with 100 ng/ml of the NK-1 receptor
antagonist.
The ability of the anti-substance P Abs and the NK-1 receptor
antagonist to inhibit neurite activation of RBLs was also observed
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FIGURE 2. A, A representative trace illustrating the sequential (neurite
first, then RBL) increase in fluorescence evoked by SV (100 pg/ml) in
SCG-RBL cocultures. B, The percentage of RBLs responding to SV stimulation (n 5 15 for both10 and 100 pg/ml SV).
The Journal of Immunology
in cocultures treated with SV. For example, the number of RBLs
in coculture responding to 10 pg/ml of SV was reduced from 67%
to 33% and 31% in the presence of anti-substance P Abs (20 mg/
ml) or the NK-1 receptor antagonist (100 ng/ml), respectively.
RBL-to-neurite communication
SCG-RBL cell cocultures were established, and, following Fluo-3
loading, the RBLs were stimulated with anti-IgE receptor Abs (antiIgE receptor Ab had no direct effect in pure SCG cultures). This
treatment resulted in the expected increase in RBL Ca21 and was
followed by an increase in neurite fluorescence (22.0 6 5.8 units
of fluorescence) (Fig. 5) with a lag time of 36.9 6 16.6 s (n 5 8).
As opposed to coculture experiments involving neurite stimulation
with SV or BK, where RBL activation was dependent on proximity
FIGURE 4. Twenty-minute pretreatment of SCG-RBL cocultures with
the NK-1 receptor antagonist, CP-99,994-1, but not the NK-2 receptor
antagonist, SR 48968, inhibited the RBL activation (i.e., increased fluorescence) in response to BK (10 nM) activation of the neurites. A, A representative tracing showing that the NK-1 receptor antagonist (NK-1 RA;
100 ng/ml) inhibits the RBL response in coculture and is without effect
upon neurite activation. This event is quantified in B (n 5 10) and is shown
to be dose-dependent. C, Addition of the NK-2 receptor antagonist (NK-2
RA; 1 mg/ml) to BK-simulated cocultures did not affect the subsequent
RBL response (n 5 7).
to a neurite, RBL activation of neurites was observed at much
greater distances, up to 160 mm.
Discussion
Considerable evidence exists for a consistent anatomical association between mast cells and nerves in tissues throughout the body
(see Ref. 4). The morphological juxtaposition of mast cells and
nerves would, by itself, be of little interest if it were not for evidence of physiological connectedness. Ag activation of mast cells
results in the release of a variety of effector molecules (e.g., arachidonic acid metabolites, 5-hydroxytryptamine, histamine, cytokines) that can modulate neuronal activity. In the reciprocal communication pathway, in vitro studies have revealed that neuronalderived messengers, such as neuropeptides, can evoke or regulate
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FIGURE 3. Twenty-minute pretreatment of SCG-RBL cocultures with
anti-SP Abs significantly inhibited the RBL activation (i.e., increased fluorescence) in response to BK (10 nM) activation of the neurites. A, A
representative tracing showing that the anti-substance P Ab (a-SP; 20 mg/
ml) inhibited the RBL response, while having no effect on the neurite
response. B, The dose-dependency of the anti-substance P Ab effect (n 5
9). C, A representative tracing illustrating that an irrelevant IgG Ab (nsIgG; 20 mg/ml) did not inhibit the RBL response in coculture with BKstimulated neurites (n 5 7).
2413
2414
FIGURE 5. Representative tracing showing that in response to anti-IgE
receptor Abs added to RBL-neurite cocultures (arrow) there is an increase
in RBL fluorescence that is followed by an activation (increased Ca21) in
the attendant neurite.
that, following activation, neuronal-derived substance P was effecting the RBLs in contact with the activated neurite.
The putative mechanism of action of substance P on mast cells
in vivo is currently a controversial issue; consequently, we tested
the ability of NK-1 and NK-2 tachykinin receptor antagonists to
modulate the RBL activation evoked in response to neurite activation. In short, and somewhat unexpectedly, only the NK-1 receptor antagonist inhibited the RBL Ca21 mobilization, while having no effect on SV- or BK-induced increases in neurite Ca21. The
effectiveness of the NK-1 receptor antagonist in this model system
was initially surprising in view of the lack of documented evidence
that NK-1 receptors are expressed on mast cells. It was feasible
that the coculture milieu and neurite contact increased expression
of an NK-1-type receptor on the RBLs. We present no data in
support of this postulate, since assessment of RBLs after coculture
showed them to be no more responsive to direct substance P application than naive RBLs. However, recent radiolabel and RTPCR studies by Cooke et al. (25) have shown that RBL-2H3 cells
do indeed express ;2225 high affinity NK-1 receptors, corroborating the findings in this study that used the same RBL cell clone.
Substance P effects upon mucosal mast cells have been thought to
occur more often as a result of interaction between the N terminus
of the neuropeptide and G proteins in the mast cell membrane,
rather than via interaction with specific tachykinin receptors (i.e.,
NK-1–3). In the light of our findings and those of Cooke et al. (25),
it is clear that the issue of receptor-mediated effects of tachykinins,
or at least substance P, on mast cells with a mucosal-type phenotype should be revisited.
In vitro studies have shown that high concentrations of substance P are necessary to cause mast cell degranulation (6), which
might suggest a limited physiological role for this interaction in
vivo. For instance, at #1 mM, substance P has been found to be
incapable of directly eliciting histamine or hexosaminidase release
from RBLs (27, 28). However, it is becoming increasingly apparent that cellular activation and selective mediator release are not
synonymous with mast cell degranulation. As only one example of
this, it has been shown that peritoneal mast cells in culture will
synthesize and release IL-6 in response to cholera toxin in the
absence of degranulation measured by histamine release (26).
Moreover, we have shown that pM doses of substance P cause
changes in ion conductance of the plasmalemma of mast cells, as
detected by electrophysiological patch clamp analysis. Furthermore, when exposed to repeated pM doses of substance P, even
after a prolonged interval, most peritoneal mast cells displayed an
increased Cl2 membrane conductance, increases in cell diameter,
and ;60% of the cells actually degranulated (13). Similarly, substance P has been shown to induce whole cell current in RBLs,
although higher doses of the tachykinin were used in that study
(29). These data, in conjunction with the present findings suggest
that low dose substance P, perhaps via intracellular Ca21 signaling
and in the absence of major degranulation, can prime or sensitize
mast cells to other stimuli. The physiological consequence of lowering the threshold to subsequent stimuli has clear implications for
neuronal-mast cell modulation of physiological events, such as the
“sensory perception” of Ag (30).
Finally, we considered mast cell-to-neurite communication. Our
data illustrate that neurites were activated in response to RBL activation caused by anti-IgE receptor Abs. Even neurites at considerable distance (#160 mm) from the mast cells were affected, and
we surmise that this is consistent with the increased amounts, and
variety of mediators, that are released upon mast cell activation/
degranulation, as compared with the lower concentrations of mediator that would be released at neuronal synapses or varicosities.
Thus, in terms of bidirectional communication, our data precisely
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mast cell activation and/or degranulation in a tissue- and speciesdependent manner. Thus, while it is widely accepted that mast
cell-nerve bidirectional communication occurs on a regular basis,
it has been unclear if an intermediary cell is required to facilitate
this functional cross-talk. Using an in vitro model, we examined
neurite-mast cell (i.e., RBL) units and have demonstrated that activation of a nerve fiber elicits an activation event (as indicated by
Ca21 mobilization) in an RBL in contact with the activated
neurite.
Electrical stimulation of nerves has been shown in vivo to cause
mast cell activation, but this is dependent upon the intensity and
type of current used, and usually occurs only with prolonged stimulation (21). Direct stimulation of the vagus nerve can result in
activation of mast cells in the dura mater (11), and Gottwald et al.
(22) have presented morphological and histochemical evidence of
mast cell activation, or even degranulation in the gut following
electrical vagal stimulation in vivo. Yet, electrical stimulation of
nerves is not always associated with mast cell activation, and vagal
stimulation in vivo was found to inhibit mast cell degranulation in
a feline model of asthma (23). In the present investigation, we
show that both BK and SV evoked neuronal activation and that
neither agent had any direct effect on RBL Ca21 flux. However,
addition of either agent to SCG-RBL cocultures resulted in RBL
activation that was always preceded by neurite activation, as
gauged by Ca21 imaging. Increased RBL Ca21 mobilization was
sustained throughout the duration of the 60-s observation period
and, in the case of BK stimulation, was invariably accompanied by
membrane ruffling and an increase in cell diameter, morphological
features associated with activation and/or subsequent degranulation. An intriguing possibility was that neurite contact influenced
the RBL’s ability to respond directly to BK or SV; however, RBLs
retrieved from cocultures were still unresponsive to direct BK or
SV application.
Following detection of substance P-like immunoreactivity in the
cultured neurites, we postulated that this tachykinin could be responsible for the RBL Ca21 flux observed after neurite activation
since: 1) substance P at high doses can cause mast cell degranulation (6); 2) lower concentrations of substance P prime mast cells
for activation at subthreshold concentrations (13); and 3) immunocytochemical characterization of nerves juxtaposed to mast cells
in situ has found many of these to be substance P-positive (24).
Addition of purified neutralizing anti-substance P Abs to the coculture milieu, dose-dependently prevented the RBL activation
elicited by BK or SV stimulation of neurites, but did not affect the
neuronal activation. This procedure did not have any direct affect
on RBLs in single culture (i.e., neurites not present), indicating
NERVE-MAST CELL INTERACTIONS
The Journal of Immunology
Acknowledgments
We thank Drs. Blennerhassett, Perdue, and Stanisz (all from McMaster
University) for technical and intellectual input.
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show that neurite or mast cell activation can result in activation of
the reciprocal cell type in the absence of any modulating or transducing effects of an intermediary cell. Clearly, afferent communication (i.e., mast cell-to-neuron) and efferent communication (neuron-to-mast cell), of which less is currently known, and the
consequent effects of such potentially reverberating circuits on local physiology may have great significance in the initiation or perpetuation of disease states, such as bronchial hyperreactivity and
asthma, idiopathic functional bowel disorders, food allergy, and
eczema.
Thus, while it has been intuitive to accept direct functional bidirectional communication within the structural confines of the
neurite-mast cell unit, in no instance before this report has actual
direct communication been demonstrated or proven between these
two cell types. Our data clearly illustrate that nerves can communicate directly with RBLs, an accepted model of a mucosal-type
mast cell (25) without the participation of an intervening cell or
cells. As a caveat, we would add that our data do not dismiss the
likelihood of other cell types in vivo modulating nerve-mast cell
communication. Nevertheless, we have shown that an activation
event in a specific neurite can result in activation in the mast celllike RBL cell in contact with the specific neurite, where Ca21
mobilization was used as an indicator of RBL activity.
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