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*
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
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 104 nerve cells onto matrigel (Becton Dickinson, Bedford, MA)-coated 35-mm diameter glass dishes. The neurons were grown in F12 culture 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-ß-D-arabinofuranoside (Ara-C, 10-6M; 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 ß-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
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 µM 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 (10-6 or 10-8 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).
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 µg/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 µg/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 µg/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 µg/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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bradykinin.
Addition of BK to SCG cultures resulted in a dose-dependent neurite
activation: 1 nM BK = 25.6 ± 5.8, 10 nM BK = 34.2
± 4.9, and 100 nM BK = 38.9 ± 7.6 units of fluorescence
intensity/neurite (n = 7; Fig. 1
, A and B), but
did not evoke an increase in fluorescence when added directly to
Fluo-3-loaded 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 ± 1.0, 4.4 ± 2.5, and 3.8 ±
1.2 s for 1, 10, and 100 nM BK, respectively (n =
9–20). Additionally, RBLs in contact with BK (10 nM)-stimulated
neurites displayed cell membrane ruffling, and cell size was increased
by 19.4 ± 2.7% (n = 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
Ca2+ signaling (data not shown).
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). Thus, 100 pg/ml or 10
pg/ml SV caused an average increase in neurite fluorescence of
30.0 ± 5.5 and 25.9 ± 3.5 units of fluorescence,
respectively (n = 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 Ca2+
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, SV-evoked neurite activation
preceded RBL activation, with a dose-dependent average lag phase of
8.3 ± 5.3 and 3.5 ± 1.9 s at concentrations of 10 and
100 pg/ml, respectively (n = 8). Concentrations of 10
and 100 pg/ml SV resulted in activation in 67 and 73% of the RBLs,
respectively (n = 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).
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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 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 = 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 abrogate the increase in RBL fluorescence evoked by anti-IgE
receptor Abs. Inclusion of the anti-substance P Abs (20 µg/ml)
(or an NK-1 receptor antagonist) did not affect the increase in RBL
fluorescence elicited directly by anti-IgE receptor Abs: 127.0
± 7.1, 124 ± 7.2, 122.3 ± 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 = 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).
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, A and B). In
contrast, the NK-2 receptor antagonist, SR48,968 (1 µg/ml), did not
affect either BK-stimulated neurite activity or the subsequent
Ca2+ 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 Ca2+
mobilization in response to 10-6 M and
10-8 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.
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RBL-to-neurite communication
SCG-RBL cell cocultures were established, and, following Fluo-3
loading, the RBLs were stimulated with anti-IgE receptor Abs
(anti-IgE receptor Ab had no direct effect in pure SCG cultures).
This treatment resulted in the expected increase in RBL
Ca2+ and was followed by an increase in neurite
fluorescence (22.0 ± 5.8 units of fluorescence) (Fig. 5) with a lag time of 36.9 ±
16.6 s (n = 8). As opposed to coculture
experiments involving neurite stimulation with SV or BK, where RBL
activation was dependent on proximity to a neurite, RBL activation of
neurites was observed at much greater distances, up to 160
µm.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 Ca2+ flux. However, addition of either agent to SCG-RBL cocultures resulted in RBL activation that was always preceded by neurite activation, as gauged by Ca2+ imaging. Increased RBL Ca2+ 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 Ca2+ 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 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 Ca2+ mobilization, while having no effect on
SV- or BK-induced increases in neurite Ca2+. 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 RT-PCR 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 µM, 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 Cl-
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 Ca2+
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 µm) 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 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 cell-like RBL cell in contact with the specific neurite, where Ca2+ mobilization was used as an indicator of RBL activity.
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Acknowledgments |
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Footnotes |
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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:
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:
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.
Received for publication January 6, 1999. Accepted for publication June 10, 1999.
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References |
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production by rat peritoneal mast cells. J. Immunol. 156:316.[Abstract]
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