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* Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands; and
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
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Introduction |
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Mast cells can be divided into various subpopulations with distinct phenotypes. Two main subsets, connective tissue type mast cells (CTMC)3 and mucosal mast cells (MMC) are recognized as distinct mast cell populations with different phenotypical and functional characteristics (9, 10). However, environmental factors such as cytokines may induce differentiation in various subsets (11). Despite their differences, both CTMC and MMC are considered to be derived from a common precursor in the bone marrow. Mast cell progenitor cells translocate from bone marrow to mucosal and connective tissues to locally undergo differentiation into mature forms. They possess a remarkable degree of plasticity, so that even apparently fully differentiated CTMC will transform their phenotype in MMC if transplanted into a mucosal environment (12). Their development and survival essentially depends on stem cell factor (SCF) and its receptor c-kit (13). Besides SCF, cytokines such as IL-3, IL-4, and IL-10 influence mast cell growth and differentiation (14), as does nerve growth factor (15, 16).
Classically, mast cells are associated with hypersensitivity reactions involving the interaction with IgE (17). However, mast cells also play a prominent role in non-IgE-mediated hypersensitivity reactions (18, 19). The sensitivity of mast cells to activation by nonimmunological stimuli such as polycationic compounds, complement proteins, superoxide anions, or neuropeptides is dependent on the subset of the mast cells examined (20).
Nonimmunological stimuli are suggested to trigger mast cell exocytosis through a receptor-independent pathway. Instead of interacting with a membrane-bound receptor, they appear to directly activate and bind to pertussis toxin-sensitive GTP-binding proteins (G proteins) through the N-terminal domain located in the inner surface of the plasma membrane (21, 22). Stimulation of G proteins will activate a signal transduction pathway, eventually leading to mast cell mediator production and release. In addition, it has been proposed that tachykinins like substance P can also induce mast cell activation via a receptor-dependent mechanism (23, 24, 25). Activation of the neurokinin (NK) receptors is dependent on the C-terminal domain of the tachykinins. C-terminal fragments of substance P cause histamine release from the mouse mast cell line MC/9 via a NK2 receptor-mediated pathway (26). Cooke et al. (25) demonstrated that RBL-2H3 cells, a rat mast cell line homologous to MMC, express the NK1 receptor for substance P on their surface. However, it is widely accepted that mast cells do not express NK receptors under normal physiological conditions. The observation that mast cell lines express NK1 receptors could represent an aberrance of these cell lines.
Most reports reveal that neuropeptides only cause degranulation at relatively high concentrations (>10-5 M). However, some researchers have shown that physiological concentrations of substance P can have biological consequences under some conditions (27). Therefore, we wondered whether NK1 receptors might be expressed on mast cells cultured from bone marrow under different conditions involving coculture with SCF and IL-4. We examined the expression of NK1 receptors on mast cells by FACS and functionally by examining the release of 
-hexosaminidase in the presence or absence of NK1 inhibitors and the NK1 receptor itself.
Previously, we have shown that substance P causes dose-dependent degranulation in bone marrow-derived mast cells (BMMC) cocultured with IL-4 and SCF for 6 days (28, 29). In this report, we have found that BMMC express functional NK1 receptors under the influence of IL-4 or SCF but are expressed in highest numbers when IL-4 and SCF are both present. The IL-4/SCF-induced up-regulation of the NK1 receptor on BMMC was associated with an increased response to substance P.
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Materials and Methods |
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Spleen cells from BALB/c mice (Charles River Breeding Laboratories, Someren, The Netherlands or Harlan, Indianapolis, IN) were cultured at a density of 2 x 106 cells/ml in RPMI 1640 medium containing 4 mM L-glutamine, 5 x 10-5 M 2-ME, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.1 mM nonessential amino acids (complete RPMI 1640) containing lectin (8 µg/ml) and placed in 75-cm2 tissue culture flasks. The cells were incubated at 37°C in a 5% CO2 humidified atmosphere. After 5–7 days, medium was collected, centrifuged for 15 min at 3200 x g, filtered through a 0.22-µm Millipore filter, and used as PWM-SCM.
Mouse bone marrow cultures
BALB/c mice, in stated experiments NK1 receptor knockout mice (a generous gift from Dr. N. Gerard, Harvard, Boston, MA), were killed by cervical dislocation and bone marrow was aseptically flushed from femurs into complete RPMI 1640. The cell suspension was washed twice in complete RPMI 1640 by centrifugation at 450 x g for 10 min and finally resuspended in complete RPMI 1640 containing 10% v/v FCS and placed in 75-cm2 tissue culture flasks at a density of 1–2 x 105 cells/ml. PWM-SCM, 20% v/v (in stated experiments IL-3 (10 ng/ml)), was added to the culture medium. Flasks were incubated at 37°C in a 5% CO2 humidified atmosphere. Cells were centrifuged and resuspended in fresh medium every week to achieve a final concentration of 1–2 x 105 cells/ml. After 3–4 wk, the BMMC were resuspended in complete RPMI 1640 and cultured with IL-4 (300 U/ml), SCF (50 ng/ml), or IL-4 plus SCF. Cytokine-treated BMMC were centrifuged and resuspended in fresh medium every 3 days to achieve a final concentration of 2 x 105 cells/ml.
FACS analysis for NK1 receptor expression
Immunofluorescence analysis was performed to determine the presence of NK1 receptors on BMMC positive for c-kit. Therefore, BMMC were monitored by double staining with the Abs against the NK1 receptor and c-kit. The anti-NK1 receptor Ab is raised against a synthetic peptide that corresponds to a 23-aa sequence (385–407) of the COOH terminus of the rat substance P receptor and is mouse and rat NK1 receptor reactive. RBL-2H3 cells were used as a positive control because these cells are known to express NK1 receptors. Cell-staining analysis was performed on BMMC cultured under different culture conditions: BMMC, IL-4-cocultured BMMC at 6 and 12 days of coculture, SCF-cocultured BMMC at 6 and 12 days of coculture, and IL-4/SCF-cocultured BMMC at 6, 12, and 18 days of coculture. Cells were harvested and resuspended at a concentration of 1 x 106 cells/ml. Cells (105) were aliquoted per tube and cells were washed using ice-cold staining buffer (PBS supplemented with 1% BSA, 0.02% EDTA, and 0.02% NaN3). Cells were centrifuged at 450 x g for 5 min at room temperature and resuspended in staining buffer with the primary rabbit Ab against either the NK1 receptor (1/100) or rabbit IgG (isotype control) was added. Biotin-conjugated c-kit or isotype control (rat IgG) was added to the designated tubes at a final concentration of 1 µg/ml and incubated for 30 min at room temperature. Next, cells were centrifuged, washed twice, and resuspended in blocking buffer (staining buffer containing 5% goat serum). FITC-conjugated goat anti-rabbit IgG was used for the detection of the NK1 receptor, PE-conjugated streptavidin was used for detection of c-kit, incubated for 30 min. Again, cells were centrifuged, washed twice, and resuspended in 500 µl of FACS buffer/tube. Cells were checked for viability with trypan blue stain before flow cytometry analysis was conducted (95–99% were live cells). Cells were analyzed using FACS WinList (version 5.0; Verity Software House, Topsham, ME).
Western blot analysis for NK 1 receptor expression
A whole cell lysate of BMMC (25 µg) or superior cervical ganglia (SCG; 4 µg) was fractionated on 12% pre-cast gels and transferred onto a nitrocellulose membrane. The membrane was washed by using TBST and blocked with 10% FBS-TBST for 1 h. Again, the membrane was washed with TBST three times for 5 min. After reacting with the primary Ab directed against the NK1 receptor (1/1,000) for 1 h, blots were washed and incubated in the presence of a goat anti-rabbit IgG conjugated with peroxidase (1/25,000). Bound enzyme was detected with an ECL system.
Activation of cells
Bone marrow cells from BALB/c mice or NK1 receptor knockout mice were cultured for 3–4 wk to develop into BMMC. At 3–4 wk, these cells were cultured with SCF and IL-4 as described above. Cells were washed twice with Tyrode’s buffer supplemented with 0.1% BSA and resuspended in Tyrode’s buffer at a density of 0.6 x 106 cells/ml. Cells (2–3 x 104) were aliquoted in 96-well plates. In stated experiments, cells were pretreated with RP67580, a specific NK1 receptor antagonist or its inactive enantiomer RP65681 (10-6 M), for 10 min. Cells were activated with different concentrations of substance P (0–200 µM) for 30 min. Total release was established by adding Nonidet P-40 to get complete lysis of cells. After a 1-h incubation of the supernatant with assay solution containing 4-methylumbelliferyl glucosaminide in 0.1 M citrate buffer (pH 4.5), the reaction was stopped by adding 0.2 M glycine buffer (pH 10.7). Fluorescence was measured using a multiwell plate reader at an emission wavelength 
= 360 nm and excitation wavelength = 450 nm. The percentage of degranulation was calculated as: ((a - b)/(t - b)] x 100, where a is the amount of -hexosaminidase released from stimulated cells, b is that released from unstimulated cells, and t is total cellular content.
Statistical analysis
Experimental results are expressed as mean and SEM. Results were tested statistically by an unpaired two-tailed Student’s t test or one-way ANOVA followed by Newman-Keuls test for comparing all pairs of groups. Analyses were performed by using GraphPad Prism (version 2.01; GraphPad, San Diego, CA). Results were considered statistically significant when p < 0.05.
Reagents
RPMI 1640, FCS, and nonessential amino acids were purchased from Life Technologies (Paisley, U.K.). Penicillin, streptomycin, L-glutamine, sodium pyruvate, 2-ME, and lectin were obtained from Sigma-Aldrich (St. Louis, MO). Murine rSCF came from PeproTech (London, U.K.). Murine rIL-4 was kindly provided by W. E. Paul (National Institutes of Health, Bethesda, MD). Substance P was purchased from Novabiochem (Laufelfingen, Switzerland). Rabbit anti-NK1 was obtained from Chemicon International (Temecula, CA) and rabbit IgG from DAKO (Missisauga, Ontario, Canada). Goat anti-rabbit-FITC was purchased from Vector Laboratories (Burlingame, CA). Streptavidin-PE conjugate was obtained from BD Biosciences (Mountain View, CA). Biotin anti-mouse CD117 (c-kit) was purchased from BD PharMingen, San Diego, CA). The 12% pre-cast gels were obtained from Bio-Rad (Hercules, CA). The BioTrace nitrocellulose membrane came from VWR Scientific (Mississauga, Ontario, Canada). Goat anti-rabbit IgG conjugated with peroxidase was obtained from Sigma-Aldrich. The ECL system came from Amersham Pharmacia Biotech (Piscataway, NJ). RP67580 and RP65681 were generous gifts from Rhône-Poulenc Rorer (Dr. C. Garrett) in France.
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Results |
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Previously, we have shown that coculture of BMMC in the presence of IL-4 and SCF increased the sensitivity to stimulation by substance P (29). To investigate whether coculture was paralleled by an increase in expression of the NK1 receptor, we analyzed binding of a NK1 receptor-specific Ab using flow cytometry. Cells were double stained for NK1 and c-kit to show the population of mature mast cells positive for the NK1 receptor. As a positive control, we first studied RBL-2H3 cells, which were known to express NK1 receptors on their surface (25). These cells indeed showed NK1 receptor expression (NK1 receptor+, 18.1%). To further confirm specificity, bone marrow from NK1 receptor knockout mice was cultured and studied for NK1 receptor expression. These BMMC were negative for the expression of the NK1 receptor (NK1 receptor+, 0%). Additionally, NK1 receptor knockout BMMC cocultured with IL-4 and SCF were also negative for the expression of the NK1 receptor (NK1 receptor+, 0%).
Next, we analyzed NK1 receptor expression on BMMC cultured in PWM-SCM or in stated experiments with IL-3-conditioned medium. On these PWM-SCM cultured cells, only 7.3% of the cells showed expression of the NK1 receptor (Fig. 1A and Table I). BMMC were now studied after 6, 12, and 18 days of treatment with IL-4 and/or SCF. These levels of NK1 receptor-positive mast cells represent significant increases compared with untreated cells. After 6 days of cytokine treatment, the percentage of cells expressing c-kit and the NK1 receptor increased significantly compared with nontreated BMMC. IL-4 by itself (Fig. 1B) showed no effect on the expression of the NK1 receptor compared with nontreated BMMC at this time of culture, whereas treatment with SCF and the combination of IL-4 and SCF displayed a significant increase compared with nontreated mast cells (Fig. 1, C and D).
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In addition, flow cytometry results were further confirmed by Western blot analysis. As shown in Fig. 3, analysis of protein lysates from BMMC revealed a prominent band identical in mobility and molecular mass to that seen in SCG. Furthermore, BMMC cocultured with IL-4 and SCF together displayed an evident increase in the amount of NK1 receptor expression over BMMC cocultured with IL-4 or SCF alone. These results are in line with the data obtained from flow cytometry.
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IL-4/SCF coculture increased substance P-induced mast cell activation
In the next set of experiments, we studied whether the increased NK1 receptor expression on BMMC was accompanied by enhanced sensitivity to stimulation by substance P. Nontreated BMMC showed only minor degranulation upon stimulation with substance P (Fig. 4). In comparison, BMMC cocultured for 6 days with SCF and IL-4 showed significant dose-dependent degranulation upon substance P stimulation (Fig. 4). A significant increase in responsiveness was seen at all concentrations of substance P used to stimulate mast cells (10–200 µM). Moreover, continued coculture with IL-4/SCF for 12 days increased the response to substance P significantly (Fig. 4). The increased responsiveness was seen at all substance P concentrations but was more marked in the lower concentration range (Fig. 4).
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We addressed whether stimulation of BMMC was NK1 receptor dependent. Therefore, IL-4/SCF BMMC cocultured for 12 days were incubated with RP67580, a specific NK1 receptor antagonist, before activation with substance P. Blockade of the NK1 receptor resulted in a significant decrease of substance P-induced -hexosaminidase release at a low concentration range (10–75 µM; Fig. 5). Stimulation at higher concentrations of substance P (100–200 µM) was not significantly affected by RP67580 (Fig. 5). The inactive enantiomer RP65681, used as a control for specificity, did not affect the dose-dependent release of -hexosaminidase (Fig. 5). Furthermore, we studied the -hexosaminidase secretion in the presence of the NK2 receptor antagonist SR48968. Blockade of the NK2 receptor did not have any effect on the substance P-induced release of -hexosaminidase (data not shown).
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In addition, BMMC were grown from NK1 receptor knockout mice and were cocultured with IL-4/SCF. At high concentrations (100–200 µM), the release of -hexosaminidase by substance P was comparable in BMMC from BALB/c mice and BMMC grown from NK1 receptor knockout mice (Fig. 6). However, NK1 receptor knockout BMMC released significantly less -hexosaminidase in the lower concentration range (0–75 µM) than did normal BMMC (Fig. 6). These results suggest that at lower concentrations of substance P, both a receptor-dependent and -independent mechanism are involved in the induction of mast cell degranulation. At higher concentrations, the contribution of the receptor-dependent pathway seems to be insignificant.
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Discussion |
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In previous work, we have shown that BMMC gained sensitivity to substance P in the presence of IL-4 and SCF (28, 29). Tsai et al. (30) showed that SCF induces proliferation of mouse mast cells in vitro and in vivo. However, optimal murine mast cell proliferation and differentiation in response to SCF may require cofactors such as IL-4 (14). IL-4 synergistically enhanced the proliferation of various mast cell lines in the presence of IL-3 or SCF (31, 32, 33, 34). In the present study, IL-4 functioned as an efficient cofactor for SCF to induce the expression of NK1 receptors on BMMC. A combination of IL-4 and SCF is sufficient to induce stable expression of the NK1 receptor on primary cultured mast cells. However, in time IL-4 and SCF alone also induced a significant increase in expression of NK1 receptors.
Mast cells may exhibit enhanced expression of NK receptors in certain pathophysiological conditions. In line with our results, it has been shown that in murine peritoneal macrophages the level of expression of the NK1 receptor can be increased following exposure to IL-4 (35). This suggests an increased expression of NK1 receptors in inflammatory conditions. In vivo, several investigators have discussed the increased expression of the NK1 receptor during inflammation. Mantyh et al. (36, 37) have shown that NK1 receptors were significantly up-regulated in inflamed tissues, on epithelium, in blood vessels, and in lymphoid accumulations. Pothoulakis et al. (38) provide evidence for increased NK1 receptor expression in the intestinal epithelium shortly after exposure to Clostridium Toxin A. Furthermore, increased levels of bronchoalveolar lavage lymphocyte mRNA encoding NK1 receptors were found in a murine model of immune inflammation in the lung (39). Up-regulation of NK1 receptors on cultured macrophages following exposure to LPS in rats and Salmonella in mice has been reported (40). However, in vivo no studies have been reported on the increased expression of the NK1 receptor on the mast cell.
SCF and IL-4 have been implicated to play an important role in the pathogenesis of inflammatory diseases, including atopic dermatitis (41, 42) and asthma (43). We hypothesize that elevated levels of IL-4 and SCF in tissues undergoing inflammation could result in the enhanced expression of NK1 receptors on inflammatory tissue resident cells such as mast cells.
Substance P can induce histamine release from several types of mast cells such as rat peritoneal mast cells, dural mast cells (44), human skin mast cells (45), and mucosal mast cells (6). Since high concentrations of substance P (micromolar) are required to induce mast cell activation, and dependent on the positively charged N-terminal amino acid residues of the substance P molecule (46, 47), the mechanism of degranulation by substance P is believed to be the result of a direct G protein activation rather than a specific receptor-mediated process (48). However, several studies support the hypothesis that NK can activate mast cells via a specific receptor-dependent pathway. Ogawa et al. (5) showed on rat peritoneal mast cells that substance P stimulated NK1 receptors to release histamine. This was significantly blocked by the NK1 receptor antagonist CP96345. Okada et al. (49) demonstrated that peritoneal mast cells of rats express functional NK1 receptors (49), but this is, so far, the only report of NK1 receptor expression on mast cells in vitro.
In this study, we show for the first time inducible expression on primary cultured mast cells. Coculture of BMMC with SCF and IL-4 results in a time-dependent increased expression of the NK1 receptor and leads to an increased sensitivity to degranulation by substance P. Several lines of evidence indicate that substance P can stimulate BMMC by a NK1 receptor-dependent route. First, a NK1 receptor antagonist was able to partially block mast cell activation by substance P. Second, BMMC cultured from NK1 receptor knockout mice show significant exocytosis but only upon exposure to high concentrations of substance P. However, since mast cell activation is not completely blocked, other pathways must also be involved. Decreased exocytosis induced by inhibition or absence of the NK1 receptor was only found at lower concentrations of substance P. At high concentrations, substance P effects mast cell degranulation by a receptor-independent way since substance P-induced degranulation was not altered in the higher concentration region. We exclude a role for the NK2 receptor in substance P-induced mast cell degranulation since blockade of the NK2 receptor did not have an effect on the activation of mast cells induced by substance P.
Several receptor-independent mechanisms for substance P action on mast cells have been suggested. Previously in our laboratory, we showed that the IL-4/SCF-induced response to substance P was partially blocked by the G protein inhibitors pertussis toxin and benzalkonium chloride (our unpublished observations). Furthermore, substance P-induced activation of BMMC is blocked by specific tyrosine kinase and protein kinase C inhibitors (our unpublished observations). These results delineate a pathway for substance P-induced secretion via G protein(s), phospholipase C, calcium, and protein kinase C and are in accordance with data in the literature. For instance, Mousli and coworkers demonstrated the involvement of pertussis toxin-sensitive G proteins and phospholipase C as targets for substance P in rat peritoneal mast cells (50) and human skin mast cells (51) without requiring substance P-specific membrane receptors.
In conclusion, the present study provides clear evidence for the inducible expression of functional NK1 receptors on BMMC in response to coculture with IL-4 and SCF. Increased expression of the NK1 receptor is accompanied by enhanced sensitivity of the mast cells to stimulation by substance P. Our data suggest that under specific conditions in vivo, such as those accompanying inflammation, mast cells could gain increased responsiveness to substance P, which may propagate and enhance neurogenic inflammatory responses. In turn, we suggest that NK1 receptor antagonists may be partially effective in minor inflammatory conditions, but are less likely to be effective when inflammation is more severe.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprints request to Dr. Hanneke P. M. van der Kleij, Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, P.O. Box 80082, 3508 TB Utrecht, The Netherlands. E-mail address: J.P.M.vanderKleij{at}Pharm.uu.nl
3 Abbreviations used in this paper: CTMC, connective tissue-type mast cell; MMC, mucosal mast cell; BMMC, bone marrow-derived mast cell; NK, neurokinin; PWM-SCM, pokeweed mitogen-stimulated spleen cell-conditioned medium; SCG, superior cervical ganglia; SCF, stem cell factor.
Received for publication August 5, 2002. Accepted for publication June 18, 2003.
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References |
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and IL-8 following stimulation with anti-IgE and other secretagogues. Exp. Dermatol. 10:312.[Medline]
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up-regulate substance P receptor expression in murine peritoneal macrophages. J. Immunol. 165:182.This article has been cited by other articles:
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  E. E. Forbes, K. Groschwitz, J. P. Abonia, E. B. Brandt, E. Cohen, C. Blanchard, R. Ahrens, L. Seidu, A. McKenzie, R. Strait, et al. IL-9- and mast cell-mediated intestinal permeability predisposes to oral antigen hypersensitivity J. Exp. Med., April 14, 2008; 205(4): 897 - 913. [Abstract] [Full Text] [PDF]
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, Nontreated BMMC; 
, IL-4/SCF-cocultured BMMC for 6 days, and 
, BMMC cocultured with IL-4 and SCF for 12 days. Results are expressed as mean ± SEM of quadruplicate samples. Significant differences (p < 0.05) between the 6-day coculture group and the nontreated group are denoted by (+). Significant differences (p < 0.05) between the 12-day coculture group and both other groups are denoted by (*).
, IL-4/SCF-cocultured BMMC pretreated with RP65681; and 