1 Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya 467, Japan; 2 National Institute of Health Sciences, Tokyo 158, Japan; and 3 Intestinal Diseases Research Program, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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Mast cell-neurite interaction serves as a model for neuroimmune interaction. We have shown that neurite-mast cell communication can occur via substance P interacting with neurokinin (NK)-1 receptors on the mucosal mast cell-like cell, the rat basophilic leukemia (RBL) cell. Neurite (murine superior cervical ganglia) and RBL cell [expressing the granule-associated antigen CD63-green fluorescent protein (GFP) conjugate] cocultures were established and stimulated with bradykinin (BK; 10 nM) or scorpion venom (SV; 10 pg/ml), both of which activate only neurites. Cell activation was assessed by confocal imaging of Ca2+ (cells preloaded with fluo 3), and analyses of RBL CD63-GFP+ granule movement were conducted. Neurite activation by BK or SV was followed by RBL Ca2+ mobilization, which was inhibited by an NK-1 receptor antagonist (NK-1 RA). Moreover, membrane ruffling was observed on RBL pseudopodial extensions in contact with the activated neurite, but not on noncontacting pseudopodia. RBL membrane ruffling was inhibited by NK-1 RA, but not NK-2 RA, and was accompanied by a significant increase in granule movement (0.13 ± 0.04 vs. 0.05 ± 0.01 µm/s) that was most evident at the point of neurite contact: many of the granules moved toward the plasmalemma. This is the first documentation of such precise (restricted to the membrane's contact site) transfer of information between nerves and mast cells that could allow for very subtle in vivo communication between these two cell types.
neuroimmunity; substance P; neurokinin-1 receptor; CD63; granule tracking
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INTRODUCTION |
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ANALYSES OF NERVE-MAST CELL INTERACTIONS have been of significant importance in unequivocally establishing the concept of bidirectional neuroimmune communication (13, 25). In addition to the anatomic association of mast cells and nerve fibers in many tissues (3, 20), numerous studies have shown that mast cell activation can be evoked by nerve stimulation or the application of neurotransmitters and that mast cell-derived mediators can influence neuronal activity (7, 18, 24). For instance, the neuropeptide substance P has been shown to cause mast cell degranulation when used at high doses, whereas exposure to picomolar concentrations of substance P primes the mast cell, lowering the degranulation stimulation threshold to a second stimulus (10). We have used an in vitro model of mast cell-nerve interaction, composed of cocultures of the mucosal mast cell-like rat basophilic leukemic (RBL) cells and neurite-sprouting murine superior cervical ganglia (5). We showed that nerve-mast cell communication did not require transduction by an intermediate cell and that the RBL cell activation response following neurite stimulation was mediated largely via substance P release and through neurokinin (NK)-1 receptors (21). Here, we sought to further examine neurite-RBL cell interactions, to determine whether neuron-induced mast cell activation is a generalized whole cell response or if there is an additional level of subtlety to the neurite-RBL cell interaction that occurs at the specific contact point between the two cells.
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MATERIALS AND METHODS |
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Nerve-RBL cell coculture.
CBA mice (Japan SLC, Shizuoka, Japan) were housed under standard
conditions and in compliance with national guidelines for the use of
animals in experimentation. Superior cervical ganglia (SCG) were
excised from newborn (<48 h old) pups, and neurite-RBL cell cocultures
were established following a methodology outlined previously (5,
21). In all experiments, RBL cells (104 cells per
culture dish) were added to 48-h-old SCG cultures and incubated at
37°C for 72 h. In the majority of cases, a single neurite was in
contact with the RBL cell, although up to three neurites were
occasionally noted in association with a single RBL cell (1 RBL cell
with 2 neurite contacts is depicted in Fig. 1); however, all of the
visualization studies focused on a single point of neurite-RBL cell
membrane contact. The RBL cells used in this study had been transfected
with a plasmid encoding CD63 (a granule-associated antigen)-green
fluorescent protein (GFP) conjugate (2, 23). Specific
neurite activation was evoked by addition of bradykinin (BK; 10 nM;
Bachem, Bubendorf, Switzerland) or scorpion venom (SV; 10 pg/ml; Sigma
Chemical) to the coculture dish. The dose of each stimulus was defined
as optimal in our earlier studies, where we also showed that the RBL
cells do not directly mobilize Ca2+ in response to these
concentrations of BK or SV in the absence of the neurites
(21).
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Analyses of neurite-RBL interaction.
After 72 h of coculture, the cells were gently rinsed (3 times)
with HEPES buffer. For Ca2+ mobilization, the cells were
incubated at 37°C for 30 min in culture medium containing 2.5 µM
fluo 3-AM (Molecular Probes, Eugene, OR), followed by three rinses in
HEPES buffer (21). Use of the CD63-GFP+ RBL
cells allowed visualization of granule movement. Subsequently, the
coculture preparations were viewed in a confocal laser scanning microscope (CLSM) (LSM-510; Zeiss, Oberkochen, Germany), BK or SV was
added to the coculture, and CD63-GFP and fluo 3 fluorescence (i.e.,
Ca2+ mobilization) were observed using excitation and
emission wavelengths of 488 and >505 nm, respectively. Images were
captured and analyzed with the Scenic Pro M7 computer analysis system
(Siemens) (21). [While the emissions from the fluo 3 and
GFP overlap, the fluo 3 was of significant magnitude and intensity that
it was readily detectable and differentiated from the background signal
due to GFP expression (see Fig. 2).] Phase-contrast images of the
cocultures were also captured.
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Statistical analysis.
Data are presented as percentages of cells responding (determined over
the different days of experimentation), expressed as means ± SE,
from n RBL cell-neurite cocultures. Data were analyzed by
Student's t-test, and P < 0.05 was
accepted as a level of statistically significant difference. A positive
ruffling response was defined as the occurrence of obvious membrane
deformities and a change in the cross section of the RBL cell
pseudopodium of 10% of the resting diameter (analyzes performed with
NIH Image software).
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RESULTS |
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Neurites establish contact with CD63-GFP RBL cells in vitro and evoke activation. After 72 h in coculture, neurites formed contacts with CD63-GFP+ RBL cells (Fig. 1) in a manner that was indistinguishable from the interaction with the parent RBL cells used in our previous study (21). Addition of BK (10 nM) to the cocultures resulted in an increase in neurite fluo 3 fluorescence within 10 s that peaked within 15 s (Fig. 2A). This neurite activation event was followed by RBL cell activation, indicated by a transient Ca2+ mobilization in the RBL cells in contact with an activated neurite. The time lag between neurite and RBL cell activation was up to 100 s, and the activation response (i.e., increased fluorescence) was observed in 48% of the cells examined (n = 40 neurite-RBL cell units; Fig. 2B). The Ca2+ response in the RBL cells was always initiated at the site of neurite contact and then moved across the cell. The increased fluorescence (i.e., Ca2+ mobilization) retuned to basal levels within 20 s (Fig. 2A). Similarly, CD63-GFP+ RBL cell-neurite cultures responded to SV (10 pg/ml) with an RBL cell Ca2+ mobilization following neurite activation in a time course that largely mirrored the BK response: 45% of the RBL cells responded (n = 20). These findings are similar to observations made with the parent, nontransfected RBL cells (21).
Pretreatment of RBL cell-neurite cocultures with the NK-1 receptor antagonist CP-99994-01 resulted in a statistically significant reduction in the number of RBL cells mobilizing Ca2+ subsequent to BK neurite activation: only 2 of 17 RBL cells observed responded, i.e., 12% (Fig. 2B; P < 0.05 compared with control).Activated neurites cause RBL cell membrane ruffling at the contact
site.
BK (10 nM) activation of the neurites resulted in subsequent RBL cell
membrane ruffling. The representative differential interference contrast image in Fig. 1b illustrates the characteristic RBL
cell membrane deformities at the site of neurite contact. These
membrane distortions occurred after ~3 min of addition of BK to the
coculture and were often still apparent 20 min after neurite
activation. This appearance suggested a local and highly specific
activation response. Next, we examined this membrane ruffling response
in CD63-GFP+ RBL cells in contact with BK-activated
neurites. A typical example of membrane ruffling of these RBL cells is
shown in Fig. 3. The phase-contrast image
in Fig. 3A, left, shows a neurite in contact with
the upper pseudopodium of an RBL cell (Fig. 3A,
right, shows the fluorescent RBL only). After BK activation
of the neurites, there was significant and obvious RBL cell membrane
ruffling, but this was restricted to the point of contact with the
neurite [Fig. 3A, compare boxes a (RBL
cell-neurite contact point) and b]. Figure 3B
illustrates the membrane ruffling at the RBL cell-neurite contact
point (a) and the RBL cell pseudopodium not in contact with
a neurite (b) over a 552-s observation period; there are significant membrane distortions only in the former instance. Overall
67% of the RBL cells examined displayed clear evidence of membrane
ruffling after BK stimulation (i.e., 10 of 15 RBL cells observed).
Addition of SV (10 pg/ml) to the RBL cell-neurite cocultures also
induced a local ruffling response in the pseudopodium of RBL cells in
contact with neurites but not in RBL cell pseudopodia that were distant
from a neurite. The time course of SV-induced membrane ruffling was
similar to that induced by BK (data not shown).
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RBL granule movements.
Use of CD63-GFP+ RBL cells allowed tracking of granule
movements following activation (i.e., Ca2+ mobilization in
this instance) in living cells with the use of CLSM. The diameter of
the fluorescent granules ranged from 0.5 to 1.5 µm. Figure
5 shows a phase-contrast image, a
fluorescence image with enumerated CD63-GFP+ granules, and
diagrams of the granules and their pattern of movement in control RBL
cell-neurite cocultures (A) and cocultures stimulated with
BK (10 nM) (B). In the absence of neurite stimulation, the RBL cell granules were largely inactive (Fig. 5Ac, note the
lack of "movement" lines from the identified granules): ~90% of
the granules did not move at all, and those that did fluctuated
randomly in the local area. In contrast, after neurite activation, we
observed increased granule movements in the contacting RBL cell, with
the most dramatic granule movements being predominantly
located adjacent to the neurite contact point. Those granules located
close to the neurite (i.e., within 10 µm of the contact point) became
highly motile and moved toward the cell plasmalemma at an average
velocity of 0.13 ± 0.04 µm/s (Fig. 5Bd;
P < 0.05 compared with nonactivated RBL cells); upward
movement of granules was also noted as some granules would leave the
focal plane of the image. Granules distant from the site of contact
were considerably less motile (0.05 ± 0.01 µm/s), and their
activity/movement patterns were, on average, largely reminiscent of the
granules in RBL cells in the nonstimulated cocultures. The most
vigorous granule movement occurred ~70-120 s after addition of
BK to the coculture (compare Fig. 5B, c and d), and this finding correlates with the Ca2+
response in the GFP+ RBL cells (Fig. 2A).
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DISCUSSION |
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The tradition of considering the nervous and immune systems as
discrete entities has been replaced with the concept of neuroimmunity, in which strictly neural or immune functions are recognized as opposite
ends of a biological spectrum. Delineation of mast cell-nerve interactions has been of primary importance in defining the neuroimmune system as an integrated system, with shared receptors and messenger molecules allowing for bidirectional communication (13).
In assessing mast cell function, a response has typically been defined in terms of degranulation (full blown or piecemeal) by measuring released histamine, products of arachidonic acid metabolism, tryptase, -hexosaminidase, or cytokines such as IL-6 and TNF-
(12). However, exposure to picomolar doses of substance P
can prime mast cells for subsequent activity (10), and
more recently, utilizing RBL cells as a model mucosa-like mast cell, we
showed that neurite activation could elicit Ca2+
mobilization in a contacting RBL cell that was inhibited by NK-1, but
not NK-2, receptor blockade (21). This study unequivocally showed the potential for neurons to direct mast cell activity in the
absence of any intermediate cell type. Extending these observations,
the current study presents data illustrative of a subtle level of
information transfer that resides at the point of membrane contact
between the neurite and RBL cell.
Using the murine SCG-RBL cell system as a model of neuron-mast cell
association, we showed bidirectional communication between the cells by
using either specific neuronal [BK or SV (21)] or mast
cell [i.e., anti-IgE antibodies (22)] activators as the
initiating stimuli. These studies depended largely on Ca2+
imaging as the index of cell activity. Surface expression of the
granule-associated antigen CD63 (2, 4) increased when basophils or RBL cells were activated with anti-IgE antibodies, and
this finding correlated with the release of histamine and -hexosaminidase, respectively (2, 9, 11). These data suggested that the expression of CD63 could be a useful surrogate of
mast cell activation, particularly at the single-cell level, where the
amount of mediators released is likely to be below the detection limits
of commercial assays. To test this possibility, we cocultured RBL cells
expressing a CD63-GFP conjugate with neurite-sprouting murine SCG. The
neurites and RBL cells formed contacts in vitro, and neural activation
by BK or SV was followed by RBL cell activation (i.e., Ca2+
mobilization). The Ca2+ response, though obvious, was not
identical to that observed in the parent RBL cells, being of a more
transitory nature (it is not unusual for GFP-transfected cells to
display slight differences compared with the parent cells).
Nevertheless, there was a clear lag phase between the neurite response
and RBL cell activation, with the latter event being inhibited by use
of an NK-1 receptor antagonist, matching the findings of our previous
investigation with the parent RBL cell line (21).
Tracking of the fluorescent granules in the RBL cells revealed a directed response to the activation stimuli received from the neurite. Before activation, the RBL cell CD63-GFP+ granules were mostly static, exhibiting only minor oscillations within the plane of focus. After activation, the velocity of the granule movement increased approximately threefold and the granules migrated toward the plasmalemma (both lateral and vertical granule movements were common); granules predominantly within 10 µm of the neurite contact site exhibited this directed movement. These observations support the concept of stimulus-secretion coupling, where Ca2+ mobilization (and likely subsequent involvement of GTP and Rho-family members) results in significant granule movement and, ultimately, exocytosis (15). Moreover, whereas granule movement was generally increased throughout the RBL cell, the most striking granule movements (in terms of speed and distance moved across the cell) were more obvious at the point of contact between the activated neurite and the RBL cell. These dramatic granule movements correlated with the lag phase in the RBL cell Ca2+ response (i.e., maximal at 70-150 s after BK was added to the coculture), suggesting that Ca2+ mobilization may be a prerequisite for enhanced granule movements.
More visually striking than the enhanced granule movement was the membrane ruffling that occurred on RBL cells associated with an activated neurite. The membrane ruffling mirrored Ca2+ mobilization and, similarly, was inhibited by blockade of NK-1, but not NK-2, receptors, underscoring the importance of substance P as a messenger in this model of neuroimmune communication. Of equal note was the fact that the membrane ruffling was restricted to the point of contact between the neurite and the RBL cell, indicating a level of sophistication in nerve-mast cell cross talk that has hitherto not been appreciated (a more global RBL cell membrane ruffling response occurred in cells treated with high- but not low-dose substance P directly). In other cell types, membrane ruffling has been associated with cytoskeletal rearrangements and signal transduction, increased motility, adhesion to extracellular matrix, and enhanced macropinocytosis (1, 6, 8, 14, 17). In earlier studies, we found that neurite-RBL cell contacts were maintained for up to 120 h in culture (end of the experiment) and that the RBL cell can move along the neurite surface toward the cell body (5). Thus we postulated that RBL cell membrane ruffling might precede a migratory response. However, observations of neurite-RBL cell units revealed no significant spatial changes 1 h after neurite activation: the RBL cell moved neither away from the neurite nor along its axonal process. These observations raise the question of whether, and under what circumstances, migratory cells such as mast cells that have made contact with a neurite and become sessile can break that contact. The precise mechanism and full biological relevance of neuron-induced mast cell membrane ruffling (and granule movement) requires extensive research efforts, the outcome of which is likely to influence our understanding of the subtlety of neuroimmune interactions.
Although we can only speculate on the biological relevance of this exquisite nerve-mast cell interaction, a number of possibilities exist. The release of mast cell-derived mediators feeding back onto the activating nerve fiber has the potential to affect the function of the nerve, altering membrane currents, sensitivity to depolarizing stimuli, or, conversely, resulting in neuron desensitization (19). Changes in neuronal activity could then impact on a number of local physiological processes such as muscle contraction and nociceptive thresholds. Indeed, analysis of colorectal biopsy specimens from some patients with inflammatory bowel disease (i.e., Crohn's disease) revealed an increased sensitivity to substance P as measured by histamine release, suggesting that the inflammation had altered nerve-mast cell interactions (16). Thus, should the precise neurite (i.e., nerve fiber)-RBL cell (i.e., mast cell) interaction described here exist in vivo, it has the potential to act as a fine-tuning mechanism in the regulation of normal homeostatic physiological and pathophysiological reactions.
In conclusion, this study supports an important role for substance P in nerve-to-mast cell communication and defines the movement of locally sited CD63+ granules toward the plasmalemma as a consequence of excitation and a possible precursor to degranulation. The demonstration that neurite activation not only causes a Ca2+ response in the RBL cell associated with the neurite but is followed by dramatic plasmalemma ruffling and enhanced granule movements at the cell-cell contact site highlights a very precise, confined, and specific means of cross talk between these cell types. The physiological relevance of this interaction needs to be defined, but clearly such intercellular communication must be important not only in health but also in the many conditions in which nerves and mast cells have been implicated, including mucosal inflammation, hypersensitivity/allergic reactions, and hyperalgesia.
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ACKNOWLEDGEMENTS |
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Technical assistance from Dr. Premsyl Bercik (McMaster University) is greatly appreciated.
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FOOTNOTES |
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This work was supported by research funds from the Ministry of Education of Japan (to M. Nakanishi) and the Canadian Institutes for Health Research Grant MT-13421 (to D. M. McKay).
Address for reprint requests and other correspondence: J. Bienenstock, Intestinal Diseases Research Program, McMaster Univ., HSC-3N21, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5 (E-mail: bienens{at}mcmaster.ca) or M. Nakanishi, Dept. of Analytical Chemistry and Biophysics, Faculty of Pharmaceutical Sciences, Nagoya City Univ., Tanabe-dori, Mizuho-ku, Nagoya 467, Japan (E-mail: mamoru{at}phar.nagoya-cu.ac.jp).
1 Supplementary material to this article (Movies 3 and 5) are available online at http://ajpcell.physiology.org/cgi/content/full/283/6/C1738/DC1.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 14, 2002;10.1152/ajpcell.00050.2002
Received 31 January 2002; accepted in final form 17 July 2002.
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