1Laboratory of Cell Biology and Histology, Department of Biomedical Sciences, 2Laboratory of Electrobiology, University of Antwerp, B-2020 Antwerp, Belgium; 3Department of Veterinary Clinical Studies, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh EH25 9RG, United Kingdom; and 4Departments of Medical Physiology and Surgery, University Medical Centre, 3584 CG Utrecht, The Netherlands
Submitted 19 December 2003 ; accepted in final form 1 March 2004
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ABSTRACT |
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calcium imaging; electrophysiology; cultured mucosal mast cells; cultured dorsal root ganglia neurons; mast cell juice
There is abundant evidence that substance P (SP) released from nerve fibers induces mast cell degranulation (9, 28, 40, 43) and that mast cell-derived mediators, such as histamine and serotonin, can influence neuronal activity (11, 12, 46). The most direct evidence available for such a bidirectional interaction has been obtained in an elegant coculture approach of rat basophilic leukemia cells (RBL) and neurons of the superior cervical ganglion (43, 44). Measurements of intracellular Ca2+ concentration ([Ca2+]i) in both cell types showed that IgE-induced degranulation of RBL cells causes activation of neurites (44) and that bradykinin-induced SP release from neurites causes NK1 receptor-mediated activation of the RBL cells (43). Remarkably, however, most of the information available about the degranulatory properties of neuropeptides has been obtained in studies using SP (9, 28, 40, 43), whereas data on the degranulatory properties of other neuropeptides, such as CGRP, are scarce (21, 49).
So far, most of the direct evidence for mast cell degranulation by neuropeptides (21, 40) has been obtained in studies on connective tissue mast cells (CTMC), which are found in connective and muscle tissues. The other type of mast cells, the mucosal mast cells (MMC), are abundantly present in the inflamed mucosa of the gastrointestinal tract (6, 39). Both mast cell types originate from the same bone marrow precursor cell, but tissue-specific signals drive them toward CTMC or MMC differentiation (26). In the mouse, two mast cell types can be distinguished (47): 1) the atypical, T cell-dependent MMCs, which contain the soluble -chymase mouse mast cell protease-1 (mMCP-I) and a limited amount of histamine and 2) the T cell-independent CTMC, which contain heparin and large amounts of histamine but no mMCP-I (3, 6). At present, no information is available about the involvement of MMC in bidirectional communication with nerve fibers.
We previously reported (6) that the parasite infection of schistosomiasis in the mouse ileum is accompanied by a mastocytosis, which is characterized by a temporal distribution of distinct mast cell phenotypes. We observed in the mucosal layer a transient increase in MMC, peaking at 8 wk postinfection. Coinciding with this mastocytosis was an increase in the density of CGRP-IR extrinsic primary afferent nerve fibers in the lamina propria of both acute and chronic infected animals (5). Although these CGRP-containing extrinsic primary afferent nerve fibers in the mouse ileum do not contain SP, MMC were found in close apposition to the dense CGRP network (5). This suggested a possible role for CGRP in the communication between MMC and the dense CGRP network, to which efferent-like functions have been ascribed (15). We hypothesize that a bidirectional communication between MMC and CGRP-IR nerve fibers is involved in regulating the inflammatory response.
The present study thus aims to determine this functional bidirectional communication between MMC and CGRP-IR extrinsic primary afferent nerve fibers in vitro. To this end, primary cultures of bone marrow-derived MMC and adult dorsal root ganglia (DRG) neurons were characterized morphologically and physiologically. Recordings of [Ca2+]i and electrophysiological measurements were used to investigate 1) the degranulation of MMC by the neuropeptide CGRP and 2) the activation of CGRP-containing DRG neurons by mast cell degranulate.
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MATERIALS AND METHODS |
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Adult male Swiss mice (Iffa Credo Belgium, Brussels, Belgium) were given food and water ad libitum and were kept in a 12:12-h light-dark cycle. All experimental procedures were approved by the local ethics committee of the University of Antwerp.
Cell Cultures
Mast cell cultures.
Bone marrow-derived mast cell cultures were made as previously described (51). Briefly, male mice at the age of 1012 wk were killed by cervical dislocation, and their femurs were removed under sterile conditions. Bone marrow was washed from the femurs using a 23-gauge needle and a 5-ml syringe filled with DMEM (GIBCO Life Technologies, Paisley, UK) containing 10% FCS (Sigma, Poole, UK), 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 µg/ml fungizone, 2 mM L-glutamine, and 1 mM sodium pyruvate (DMEM/FCS). A single cell suspension was obtained by passing the material three times through a 19-gauge needle, followed by filtration through sterile lens tissue. The cell suspension was then centrifuged at 230 g during 7 min at room temperature (RT). After resuspension in 10 ml DMEM/FCS, the cells were counted in a hemocytometer (x20 magnification) using 0.2% Nigrosin (Sigma, St. Louis, MO) exclusion to measure viability. Cell cultures were set up in a humid 5% CO2 incubator at 37°C using 162-cm2 flat-bottomed flasks at 5 x 105 cells/ml in DMEM/FCS containing 50 ng/ml recombinant murine stem cell factor (SCF; Peprotech, London, UK), 5 ng/ml recombinant mouse IL (rmIL)-9 (R&D Systems, Abingdon, UK), 1 ng/ml rmIL-3 (R&D Systems), and 1 ng/ml recombinant human transforming growth factor (TGF)-1 (Sigma). This combination of cytokines is referred to as TI3S and is known to result in high MMC viability (93%) and mMCP-I concentrations (1,250 ng/ml) (51). Cultures were maintained for 9 days and were fed at 2- to 3-day intervals.
DRG neuron cultures. Adult mouse DRG neurons were isolated as previously described (33). First, animals were anesthetized by an overdose of ether and bled from the abdominal aorta. Next, the spinal cord was isolated and the ganglia dissected using microforceps. After removal of connective tissue, the spinal roots were cut close to the ganglia, which were then enzymatically digested with collagenase (5 mg/ml, 45 min, RT; Boehringer-Roche, Mannheim, Germany) followed by incubation with trypsin (GIBCO) and collagenase (each 2.5 mg/ml, 30 min, RT). The ganglia were then suspended in DMEM containing 10% FCS, collected by centrifugation, and mechanically dissociated using glass pipettes with decreasing tip diameters. The resulting cell suspensions were plated on serum-coated petri dishes to allow the nonneuronal cells to adhere. This purification is not absolute and gives rise to fibroblasts in the culture of DRG neurons. After 90 min, the neurons in suspension were collected and plated onto poly-L-lysine-coated petri dishes with glass bottoms (MatTek, Ashland, MA). Cultures were maintained for 34 days (37°C) and were fed at 2-day intervals.
ELISA Measurements
Chymase (mMCP-I). MMC supernatants was obtained before feeding cultures and stored at 80°C until further processing. The concentration of mMCP-I in culture supernatants was assayed by a sandwich-based ELISA technique using a monoclonal rat anti-mMCP-I capture antibody (5 µg/ml) and a polyclonal sheep anti-mMCP-I detection antibody (2 µg/ml) as previously described (19, 39, 51). Bound antibody was visualized using a biotinylated donkey anti-sheep IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Next, incubation with extravidin-horseradish peroxidase (Sigma) and subsequent development with 3,3',5,5'-tetra-methylbenzidine substrate (KPL, Gaithersburg, MD) resulted in a blue color. The reaction was stopped with 0.18 M H2SO4, and the optical density was measured on an ELISA plate reader (ELX-808UI, Bio-Tek Instruments, Winooski, VT) at a wavelength of 450 nm.
Serotonin and histamine. After mast cell degranulation with compound 48/80 (for details, see Drugs), histamine and serotonin analyses of the degranulate were performed with previously evaluated (18, 23) commercially available, highly specific ELISA kits (IBL, Hamburg, Germany). All experiments were performed according to the manufacturer's guidelines. Final concentrations were expressed micromolarly.
Tissue Preparation and Immunocytochemistry
Cells from the bone marrow-derived mast cell culture were washed three times and resuspended in PBS before being loaded onto Marienfeld Adhesion Slides (Paul Marienfeld, Lauda-Königshofen, Germany) according to the manufacturer's guidelines. Bound cells were fixed in modified Bouin's fluid [95% saturated picric acid, 5% concentrated (37%) formaldehyde, and 2.5% glacial acetic acid] for 10 min at RT, permeabilized in absolute methanol for 10 min at RT, and stored in 70% ethanol at 4°C until further processing for immunocytochemistry.
Cells from the primary culture of DRG neurons were fixed with 4% paraformaldehyde for 10 min at RT on the poly-L-lysine-coated glass bottom of the petri dish. Subsequently, they were rinsed three times and kept in PBS until further processing for immunocytochemistry.
All incubations were performed at RT. The primary and secondary antibodies (Table 1) were diluted in PBS containing 10% normal goat serum, 0.01% bovine serum albumin, 0.05% thimerosal, and 0.01% sodium azide (PBS). To block nonspecific immunoglobulin interactions and to enhance permeability, the fixed cells were immersed in the supplemented PBS solution to which 1% Triton X-100 was added. Next, they were incubated for 3 h with a primary antibody. Subsequently, after being rinsed in PBS, the tissue was incubated with an appropriate secondary antibody for 1 h. For negative controls, primary antisera were omitted in the protocol. The specificity of the commercial antibodies was tested by performing immunoblotting and preabsorption tests.
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A volume of 10 ml of a 9-day-old culture of both stimulated [compound 48/80 (C48/80); see Drugs] and nonstimulated MMC was centrifuged at 250 g for 7 min. The supernatants were removed, and the pellet was fixed with 0.1 M cacodylate buffered 2.5% glutaraldehyde (pH 7.4). Subsequently, the pellet was broken into small pieces and postfixed in 0.1 M cacodylate buffered 1% OsO4, followed by dehydration in acetone before embedding in Durcupan. Subsequently, after 2 days, 50-nm-thin sections were cut and contrasted with 2% uranylacetate and Reynolds solution for 10 min each. The sections were then analyzed at a primary magnification of x6,600 in a transmission electron microscope (CM10; Philips, Eindhoven, The Netherlands).
Drugs
C48/80, rat -CGRP, SP, pertussis toxin (PT), and TTX were purchased from Sigma. Neurobiotin was obtained from Vector Laboratories (Burlingame, CA). Capsaicin was purchased from Fluka (Buchs, Switzerland). Cell culture medium and FCS were supplied by GIBCO-BRL. C48/80, CGRP, SP, and PT were stored as frozen aliquots before use. TTX, neurobiotin, and capsaicin were freshly made before each experiment.
Preparation of MMC Juice
Nine-day-old cultured MMC (5 x 105 cells in 100 µl DMEM F-12 buffer; GIBCO) were treated with C48/80 (10 µg/ml) for 1 h at 37°C. After centrifugation at 250 g during 5 min, the supernatants (MMC juice) were carefully removed and stored at RT for immediate experimental use.
Optical Recording of Cytosolic [Ca2+]i
Calcium mobilization was used as an index of cellular activation and was assessed by confocal laser scanning microscopy (CLSM). Dishes with cells (for details, see below) were incubated with the Ca2+ indicator dye Fluo-4 AM (1 µM; Molecular Probes, Eugene, OR) and 0.005% of the detergents Pluronic F-127 (Sigma) in carbogenated (5% O2-95% CO2) DMEM F-12 buffer (GIBCO) for 40 min at RT. Approximately 2030 min after stopping dye loading by refreshing the buffer, the dishes were transferred to an inverted microscope (Axiovert 100M; Zeiss) equipped with a x25 water immersion objective (Zeiss; Plan-neofluar; numerical aperture 0.8) in a CLSM system (Zeiss; LSM-510). Digital images (size 368.5 x 368.5 µm) were recorded (usually during 240 s) at RT with a spatial resolution of 256 x 256 pixels and a temporal resolution of five images per second. The 488-nm argon laser line (200 mW) was used to excite Fluo-4 fluorescence in the cells, which was measured using a long-pass 505-nm filter. Laser illumination intensity was kept to a minimum (max 1% of laser output) to avoid phototoxicity and photobleaching. Calcium signals of the cultured DRG neurons were recorded in the culture dishes containing 100 µl buffer. For recordings of MMC (day 9 of culture), 5 x 105 MMC (in 100 µl of buffer) were seeded onto the poly-L-lysine-coated bottom of a well (diameter 10 mm) in a 35-mm-diameter culture dish (MatTek, Ashland, MA) and were then left
30 min to settle to the bottom. All fluorescence measurements were made from subconfluent areas of the dishes, enabling the ready identification of individual cells. To measure cellular calcium responses to application of CGRP, SP, C48/80, and mast cell juice, these compounds were dissolved in DMEM-F12 buffer and applied directly in the bath as a small drop (10 µl) from a micropipette. The location of the pipette tip was at least 500 µm from the nearest cells to avoid pressure-induced stretching of cell membranes. Because the culture dishes with DRG neurons also contained some fibroblasts, immediately following each experiment, all DRG neurons within the image were identified by visual inspection using differential interference contrast. Criteria used were shape, diameter, and thickness of the soma, presence of neurites, and neurite size.
Image data were analyzed off-line using the Zeiss LSM510 analyzing software V2.53. A selected image in each image set was used as a template for designating each cell as a region of interest. Because Fluo-4 is a single-wavelength indicator, it was not possible to apply the ratiometric method for quantitative determination of [Ca2+]i. Therefore, data were normalized with respect to the mean fluorescence intensity (Fo) during the first 510 s of recording. Temporal fluorescence intensity of the dye was divided by Fo. These relative fluorescence (RF) values represent integrated [Ca2+]i. The RF values within each region of interest were plotted as a function of time. To determine the lag time of responses to application of compounds, we measured the time between drug application (as recorded by computer) until the first noticeable change in RF in the plots. The amplitude of the transient responses of MMC to application of neuropeptides (see Fig. 6) and of the DRG neurons to mast cell juice (see Fig. 7) was quantified as the highest RF level reached during the measuring period. Given the individual differences between cell responses, the amplitude of the complex responses of MMC to application of C48/80 (see Fig. 4) was also quantified as the maximum RF reached in the plots.
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Dishes containing a culture of DRG neurons were placed on the stage of an inverted microscope (Diaphot; Nikon, Tokyo, Japan) and were continuously superfused (10 ml/min; RT) with oxygenated Krebs-Ringer solution of the following composition (in mM): 118 NaCl, 4.75 KCl, 2.54 CaCl2·2H2O, 1.2 MgSO4·7H2O, 1 NaH2PO4·2H2O, 25 NaHCO3, and 11.1 glucose. Intracellular recordings were made with borosilicate glass microelectrodes (1-mm outer diameter; Clarc Electromedical Instruments, Reading, UK) pulled on a P-97 Brown-Flaming micropipette puller (Sutter Instrument, Novato, CA). The electrodes were backfilled with 1 M KCl containing 2% neurobiotin (resistance 60100 M). Potentials were recorded with an electrometer (Axoclamp 2A; Axon Instruments, Foster City, CA) through which also rectangular current pulses (generated using pClamp 6.0.2; Axon Instruments) could be injected. After amplification and low-pass filtering (3 kHz), each signal was digitized using a Labmaster TL-1 DMA Interface (Axon Instruments) at a sample rate of 5 kHz and displayed and stored on PC. Electrophysiological measurements were made after allowing the voltage signal to stabilize for a few minutes, without applying intracellular direct current. The input resistance of the neurons was estimated by the responses to hyperpolarizing current pulses of variable amplitude (0.05 to 0.3 nA). Durations of action potentials were measured as half widths, i.e., the time interval between the point on the upstroke at which the amplitude is halfway between the membrane resting potential and the maximum potential, and the equivalent point on the downstroke.
The effect of TTX (2 µM) on the action potential was determined by adding TTX to the superfusion solution for 510 min. Capsaicin (106 M in pipette) was applied in the vicinity of the impaled neuron by pressure-pulse ejection (50 ms) using a Picospritzer II (General Valve, Fairfield, NJ). Mast cell juice (see Preparation of Mast Cell Juice) was applied by consecutive (5-s intervals) pressure-pulse ejections with increasing duration (50550 ms). Data analysis was performed on a computer (pClamp 6.0.2; Excel 97, Microsoft; Sigmaplot 5.0, SPSS Sciences).
During electrophysiological experiments, impaled neurons were iontophoretically filled with neurobiotin by passing depolarizing current pulses (0.51 nA, 100- to 500-ms duration) through the recording electrode. To reveal the presence of neurobiotin in the impaled neurons, the cultures were incubated with streptavidin coupled to Cy3 (1:4,000; Jackson ImmunoResearch Laboratories) after fixation (see Tissue Preparation and Immunocytochemistry).
Statistics
For quantification of mMCP-I levels in culture (n = 4) supernatants, median mMCP-I concentrations were compared using the nonparametric Mann-Whitney U-test.
The responses to stimulation of MMC with C48/80, CGRP, and SP were analyzed via an ANOVA test followed by a Bonferroni test to check for differences between groups when comparing the number of responding MMC and amplitudes (RF) of the responses. For all experiments, these statistical tests revealed no differences among dishes within one culture (usually 3 dishes per culture). Subsequently, results obtained on the dishes within one culture were pooled, and statistical analysis was performed among the different cultures (usually 3 cultures per experiment). Because no differences were observed, the results of all cultures were pooled, resulting in mean values for the percentage of MMC responding and the response amplitude. Stimulation of DRG neurons with MMC juice was analyzed similarly: no significant differences were found, and therefore, all cultures (n = 5) were pooled, resulting in a mean value for response amplitude. The same statistical test was performed for neurons activated by C48/80-induced MMC degranulation (n = 2). Finally, an unpaired t-test with Welch correction was used to compare the mean lag time and mean amplitude of directly (by MMC juice) and indirectly activated (by C48/80-induced MMC degranulation) DRG neurons.
All values are expressed as means ± SD. Data were considered statistically significant when P < 0.05.
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RESULTS |
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MMC cultures.
The bone marrow-derived MMC cultures (n = 20) were maintained for 9 days. Cell viability was decreased by days 2 and 4 (90 ± 0.8%; n = 3 cultures) but restored at day 9 (96 ± 0.3%; P < 0.05). The concentration of mMCP-I (a marker for MMC) in culture supernatants was 176 ± 31 ng/ml (n = 3 cultures) on day 4 and increased to 1,170 ± 50 ng/ml on day 9 (P < 0.05). Our results are in accordance with previous observations showing that addition of SCF, TGF-, IL-3, and IL-9 to the culture medium results in differentiating mast cell-like phenotypes and that this quartet of cytokines has good results on MMC viability (93%) and mMCP-I concentration (1,250 ng/ml) in differentiated MMC (51).
Leishman's staining (Fig. 1A) clearly showed the presence of granules in the cytoplasm of MMC. Immunocytochemical staining for mMCP-I (Fig. 1B) demonstrated that this protease is present in the granules, and electron microscopic (EM) analysis further revealed densely packed granules in the cytoplasm of MMC (Fig. 1C).
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DRG neuron cultures.
After 2 days in culture, the DRG neurons exhibited outgrowths of neurites that form contacts with each other (Fig. 1E) and with other neurons over relatively long distances. Immunocytochemical double staining for protein gene product (PGP) 9.5 (labels all neurons) (48) and CGRP (specifically labels primary afferent, ileum-projecting DRG neurons in mice) (5) revealed that 40% of the cultured DRG neurons were CGRP immunoreactive (IR; Fig. 1, FH). Electrophysiological experiments confirmed that two populations of DRG neurons, each having specific electrophysiological properties, can be distinguished. One population of neurons had short-duration action potentials (1.1 ± 0.7 ms; n = 11) with no shoulder on the falling phase (Fig. 3Aa). These cells displayed TTX-sensitive action potentials (2 µM; n = 9; Fig. 3Ab) and did not respond to pressure-pulse application of capsaicin (106 M; n = 5; Fig. 3Ac). The other population of neurons exhibited long-duration action potentials (2.8 ± 1.1 ms; n = 27) with a shoulder on the falling phase (Fig. 3Ba). In the latter neurons, the action potentials were resistant to TTX (2 µM; n = 8; Fig. 3Bb) and the cells depolarized (34 ± 11 mV) and reached firing threshold in response to pressure-pulse application of capsaicin (106 M; n = 5; Fig. 3Bc). On the basis of these electrophysiological properties, this population could be identified as nociceptive afferent neurons (27). Moreover, immunocytochemical analysis of the impaled neurons revealed that the neurons belonging to this population were immunoreactive for CGRP (Fig. 3C). This finding is in accordance with earlier morphological observations that capsaicin treatment results in loss of CGRP-IR (5). These results validate the cultured neurons and identify part of the cultured neurons used in this study as ileum-projecting, extrinsic primary afferent, CGRP-containing neurons.
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On MMC loaded with Fluo-4, we measured the [Ca2+]i response to bath application of C48/80 as a reference and of the neuropeptides CGRP and SP. Calcium images of MMC (day 9 of culture) were measured (5 images/s) during a period of 34 min. Application of the secretagogue C48/80 (10 µg/ml) (20, 52) induced a complex increase in [Ca2+]i in the MMC. A typical example of sequential Fluo-4 fluorescence images depicting the response of hundreds of MMC to C48/80 in a single microscopic field is shown in Fig. 4A. The regions of interest defined for each individual MMC in the field were used to calculate the RF intensity vs. time for all MMC. The calcium signals of three representative MMC are depicted in Fig. 4B. These signals show that the application of C48/80 induced, after a considerable lag period, an abrupt oscillatory increase in [Ca2+]i. After a few oscillatory cycles, the calcium signal settled to a temporarily sustained elevated level and then usually started to decrease slowly. These properties are considered characteristic of a "rapid-release" degranulatory response of mast cells (32, 52).
The percentage of responding MMC was determined from the RF traces of all MMC. Furthermore, the lag time between the application of C48/80 and the first noticeable rise in [Ca2+]i and the maximum [Ca2+]i level reached (RF) were quantified. The differences in amplitude and pattern of the Ca oscillations were not analyzed. Of all MMC to which C48/80 was applied (see MATERIALS AND METHODS for details on pooled experiments), 56 ± 1% (134/260 cells; 3 cultures) responded to C48/80 with an increase in [Ca2+]i. The mean lag time of those MMC was found to be 47 ± 21 s. The distribution of the lag times is depicted in Fig. 5. The wide range of lag times (10100 s) is in good accordance with earlier reports on mast cell degranulation (31, 44). The maximum [Ca2+]i level reached was 6.5 ± 2.
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To determine whether the effect of CGRP was mediated by Gi proteins, MMC were pretreated with PT (2 µg/ml) for 1, 2, and 3 h before determining their response to CGRP stimulation. When compared with control conditions (15 ± 6%; 66/599 cells), a marked decrease in the percentage of MMC responding to CGRP was observed after treatment with PT for 2 (5 ± 2%; 16/345 cells; P < 0.05) and 3 h (1 ± 2%; 4/1,297 cells; P < 0.001). With respect to the amplitude (RF; control: 2.7 ± 0.3; PT 3 h: 2.9 ± 0.7) and the lag period of the responses, no effect of PT treatment was observed. These results clearly indicate that the effect of CGRP was mediated by PT-sensitive Gi proteins, which is in accordance with literature on connective tissue mast cells (8, 29).
MMC Juice Induces Increase in [Ca2+]i and Depolarization of DRG Neurons
The [Ca2+]i response to bath application of MMC juice was measured on cultured DRG neurons loaded with Fluo-4. This MMC juice was prepared from the supernatant of C48/80-activated mast cell cultures (see MATERIALS AND METHODS) and undoubtedly contained a large variety of mediators, such as histamine, serotonin, neutral proteases, cytokines, and chemokines (24). On the basis of the measurements in MMC juice (ELISA; see ELISA Measurements), the final bath concentrations of histamine and serotonin were estimated at 0.6 µM each. MMC juice obtained by CGRP or SP stimulation was not used to avoid direct activation of DRG neurons by these neuropeptides.
A typical example of sequential Fluo-4 fluorescence images demonstrating the response of DRG neurons to MMC juice is depicted in Fig. 7A. Application of MMC juice induced in the soma of DRG neurons a rapid increase in [Ca2+]i, followed by a slow exponential decrease toward the baseline. The [Ca2+]i responses of the DRG somata to MMC juice were accompanied by comparable increases in [Ca2+]i in their neurites. Only the soma responses were analyzed. The calcium signals of four representative DRG neurons are depicted in Fig. 7B. We found that all (57/57; 5 cultures) DRG neurons responded to MMC juice application with a marked increase in [Ca2+]i. The mean amplitude (RF) of the response was 1.9 ± 0.4 (n = 5), and the mean lag time was 5 ± 2 s. The distribution of the lag times is shown in Fig. 8A. Activation of DRG neurons by MMC juice was often accompanied by a [Ca2+]i response of the (Fluo-4 loaded) fibroblasts present in the culture dish (example in Fig. 7A). The mean lag period of the fibroblast response was found to be 6 ± 1 s (50 fibroblasts; n = 3). Because this value is not different (P = 0.86) from that of the DRG neurons (5 s), it seems unlikely that DRG neurons were indirectly activated by compounds released from previously activated fibroblasts. The MMC juice used for activation of DRG neurons also contained C48/80. Control experiments showed that C48/80 (10 µg/ml) and MMC culture medium (not shown) had no effect on [Ca2+]i of DRG neurons (n = 3 cultures). These results demonstrate that application of MMC juice causes a fast increase in [Ca2+]i in DRG neurons, indicating neural activation.
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DISCUSSION |
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It is important to emphasize that the bone marrow cells growing in the presence of IL-3 and -9, SCF, and TGF-1 are true homologs of the MMC population that is recruited to parasitized intestine, because the cells contain the abundant granule chymases mMCP-1 and -2 (34) and express the integrin
E
7 (51). Both of these chymases are uniquely present in the MMC population and are not expressed in CTMC (13, 39). Similarly, expression of the integrin
E
7 is characteristic of cells close to, or within, mucosal epithelia (51). These in vitro correlates, therefore, establish the basis for using this model to describe neuronal mast cell interactions in the gut.
MMC Degranulation by C48/80
Studies with fluorescent indicator dyes have unequivocally shown that the increase in [Ca2+]i observed in CTMC occurs concomitantly with secretion (31) and that this increase is a necessary step in the process of degranulation (14, 16). The results of our calcium measurements on Fluo-4-loaded MMC clearly demonstrate a rise in [Ca2+]i in MMC activated by the mast cell degranulator C48/80. The EM studies and mMCP-I measurements showed that application of C48/80 to MMC induced degranulation. Our results reveal that the C48/80-evoked increase in [Ca2+]i in MMC is accompanied by a release of mediators (i.e., histamine and serotonin) and that degranulation takes place, although this may not be a complete degranulation in all cases (31).
The dose-response curve for C48/80 shows an effective concentration range, which is comparable with that necessary for degranulation of peritoneal mast cells (29). In our experiments, C48/80 was used in a concentration (10 µg/ml; EC50) that evokes in CTMC a rapid, anaphylactic shock-type, noncytolytic degranulation by which the mast cells rapidly extrude materials to the cell exterior (7, 52). The lag time for the increase in [Ca2+]i after C48/80 application was found to be 47 s, which is in line with previous reports on antigenic degranulation of RBL cells [38 s (30); range 1050 s (44)]. The variety of oscillatory activation patterns observed in C48/80-stimulated MMC corresponds with the oscillations reported in RBL studies [Ca2+ fingerprinting (25, 30)]. Although the mechanism underlying the oscillations in [Ca2+]i was not investigated in MMC, it seems most likely that C48/80 induces a biphasic increase in [Ca2+]i in MMC as was reported for the RBL cells (30) and that fluctuations in the filling state of the internal Ca2+ stores are involved in these Ca2+ oscillations (30). Because only 57% of the MMC in the culture dishes reacted to C48/80 stimulation, probably not all the cultured mast cells were sufficiently mature to respond. The low relative maturity of part of the mast cells or of their secretory granules (36) is in agreement with our observation that all MMC responding to C48/80 reached relatively high RF values (>4), whereas the other MMC showed no rise in [Ca2+]i at all.
MMC degranulation by SP and CGRP
Our finding that application of CGRP (107 M) or SP (107 M) to MMC evokes a rise in [Ca2+]i led us to conclude that CGRP and SP are able to degranulate MMC. This conclusion is in accordance with measurements of SP-induced histamine secretion from MMC (40); however, CGRP-induced degranulation of MMC has not been reported before. Almost all information on degranulation of mast cells by SP (9, 21, 37, 40, 49) or CGRP (37, 49) originates from studies on CTMC, a functionally different type of mast cells (13, 26). The working concentration of CGRP and SP (107 M), based on the dose-response curves, is in line with previous reports on CTMC (40, 49), indicating that in vitro, relatively high doses of neuropeptides are required to induce mast cell degranulation. The concentrations of CGRP and SP were chosen in such a way that 50% degranulation was expected compared with C48/80, as was actually observed during the calcium experiments. Low concentrations of neuropeptides (i.e., SP) are able to prime mast cells for activation at picomolar concentrations (17, 43). Thus, under in vivo conditions, degranulation of mast cells is most likely induced by lower concentrations than those used in the present study.
Unlike C48/80 stimulation, stimulation with CGRP and SP did not result in Ca2+ oscillations, which led to the interpretation that the neuropeptides do cause a release of mediators from MMC (17, 43), rather than an anaphylactic shock-type degranulation. C48/80 is known to be able to directly activate intracellular Gi proteins in CTMC by a mechanism referred to as the peptidergic pathway of mast cell activation (8, 29). This activation pathway involves membrane-assisted receptor-independent stimulation of Gi-like proteins and appears to be nonspecific in that it can also be used by neuropeptides, such as SP (4). Our finding that PT, a blocker of Gi proteins, completely inhibits the MMC response to CGRP, clearly demonstrates that the effect of CGRP is Gi protein mediated. Possible differences in the mechanism by which neuropeptides and C48/80 are translocated to the intracellular environment of the mast cell might explain why the lag time for neuropeptides (CGRP, 33 s; SP, 21 s) is smaller than that for C48/80 (47 s). Given the reported receptor-mediated degranulation of CTMC by neuropeptides (43, 45), it should be mentioned that our experiments do not exclude the possibility that CGRP receptors are present on the MMC surface.
Activation of DRG Neurons by Mast Cell Juice
MMC juice was shown to activate all functional types of DRG neurons, as indicated by an increase in [Ca2+]i. The time course and shape of the calcium responses of DRG neurons to MMC juice are comparable with those of the activating depolarization induced by inflammatory mediators, such as histamine (12) and serotonin (11) in enteric neurons. Our coexperimental setup with loaded DRG neurons and unloaded MMC disclosed that C48/80-mediated MMC degranulation was able to activate DRG neurons. The duration of the neural calcium responses obtained by this MMC-mediated activation was much longer (>4 min) than that obtained by direct activation of DRG neurons by MMC juice (100 s). Accordingly, the lag times of the neural responses to indirect activation were much longer (25 s) than that measured after application of MMC juice (5 s). This finding is quantitatively in accordance with our observation that >90% of the MMC show a lag time for C48/80-induced degranulation >20 s (Fig. 5). In this study, our particular interest was in the communication between MMC and the CGRP-containing extrinsic afferent neurites in the mouse ileum. The results of the electrophysiological experiments on identified (see RESULTS) neurons unequivocally demonstrate that MMC juice activates this particular population of ileum-innervating afferents.
Bidirectional communication between MMCs and DRG neurons. Intestinal schistosomiasis in the mouse ileum is accompanied by a mastocytosis and a concomitant increase in density of CGRP-IR extrinsic primary afferent nerve fibers (5, 6). The observed close apposition of MMC to the neural network (5) led to the hypothesis that a bidirectional communication between MMC and CGRP-IR nerve fibers is involved in the regulation of the inflammatory response. CGRP has chemoattractive properties (10) and is thought to be implicated in the induction of mastocytosis. The finding that CGRP is also able to directly activate MMC suggests a functional importance of CGRP as a neural messenger in the signal pathway from these particular afferent neurites to the MMC. Activation of the afferent neurites by MMC degranulate confirms the functional importance of the mast cell's inflammatory mediators in the signal pathway from MMC to the afferent nerve fibers.
In conclusion, the present study has provided evidence that MMC in culture are activated and degranulate in response to stimulation with C48/80, CGRP, and SP. In addition, MMC degranulate is able to activate DRG neurons. Taken together, these findings confirm our hypothesis that MMC activation and degranulation occur as a direct response to the release of CGRP from extrinsic primary afferent nerve fibers and that extrinsic primary afferent neurites can be activated by mast cell degranulatory compounds. More generally, our results provide support for the notion that mast cells and sensory nerve fibers are important neuroimmune components in axon-reflex pathways involved in tissue defense against injury and noxious stimuli (2, 41, 42).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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