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(Journal of Leukocyte Biology. 2002;71:829-836.)
© 2002 by Society for Leukocyte Biology

Endothelins regulate mediator production of rat tissue-cultured mucosal mast cells. Up-regulation of Th1 and inhibition of Th2 cytokines

Martin Coulombe*, Bruno Battistini*, Jana Stankova{dagger}, Philippe Pouliot* and Elyse Y. Bissonnette*

* Centre de Recherche, Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie, Université Laval, Québec, Canada; and
{dagger} Immunology Division, Université de Sherbrooke, Quebec, Canada

Correspondence: Dr. Elyse Bissonnette, Hôpital Laval, 2725, Chemin Sainte-Foy, Sainte-Foy, Quebec, Canada G1V 4G5. E-mail: elyse.bissonnette{at}med.ulaval.ca


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ABSTRACT
 
Mast cells have been shown to produce endothelin-1 (ET-1) and to express ET receptors. Thus, we postulated that ETs modulate mast cell mediator production in an autocrine manner. Rat tissue-cultured mast cells (RCMC-1) were incubated with exogenous ET-1 or ET-3, and ß-hexosaminidase release and TNF, IL-4, IL-10, IL-12, IL-13, macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), and nitric oxide (NO) production were investigated. ET-1 and -3 induced the release of ß-hexosaminidase and TNF and of mRNA expression. An antagonist of the ETB receptor subtype abrogated ET-stimulated TNF release, although ETA and ETB receptors have been identified by immunocytochemistry. It is interesting that ET-1 and ET-3 inhibited (25–30%) mRNA expression of Th2-type cytokines (IL-4, IL-10, and IL-13) and increased IL-12 release (39% and 41%, respectively) without affecting MIP-1{alpha} and NO production. Thus, our data suggest that ETs may play an important role in modulating the cytokine network by regulating Th1/Th2 cytokine production by mast cells.

Key Words: ET receptors • nitric oxide • TNF • ß-hexosaminidase • interleukin • macrophage inflammatory protein-1{alpha}


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INTRODUCTION
 
The endothelins (ETs: ET-1, ET-2, and ET-3) constitute a family of three 21-amino acid isopeptides [1 , 2 ]. These related peptides are characterized by two disulfide bridges and six conserved amino acid residues at the COOH-terminus. ET-1 differs from ET-2 by only two amino acids and from ET-3 by six amino acids. Each ET peptide is encoded by a distinct gene that codes for a precursor peptide, the preproendothelin [2 ]. Two types of ET receptors, ETA and ETB, have been cloned and characterized in mammals [3 , 4 ]. ETA binds ET-1 and ET-2 more efficiently than ET-3, and ETB shows similar affinity for all three ETs.

In the lung, ETs are produced by numerous cell types including tissue macrophages, endothelial, and epithelial cells [5 ]. Increasing evidence suggests important roles of ETs in numerous pulmonary diseases [6 , 7 ] including asthma [8 , 9 ]. ET-1 mimics several of the features of asthma such as bronchospasm, airway remodeling, mucus secretion, airway edema, and hyperreactivity [8 ]. Increased ET-1 and ET-3 levels have been shown in bronchoalveolar lavage fluid and serum of asthmatic patients compared with normal subjects, with evidence of a correlation between ET amounts and disease severity [9 , 10 ]. Furthermore, plasma and sputum levels of ET-1 are increased in allergic subjects during the early- and late-phase reactions after allergen challenge [11 ]. Although ET-1 seems to be implicated in the pathophysiology of asthma [8 ], its role in the immune response remains unclear.

Mast cells are major effector cells in allergic diseases such as asthma [12 ]. During the last decade, mast cells have been identified as an important source of multifunctional cytokines that play an important role in inflammation and allergic responses [13 , 14 ]. Although mast cells are well known to produce T-helper cell type 2 (Th2) cytokines as seen in asthma, they can also produce Th1-type cytokines, showing their functional diversity [15 ]. Furthermore, different populations of mast cells have been shown to produce ET-1 and to express ETA and/or ETB receptors depending on tissue and species [16 17 18 19 ]. Murine bone marrow-derived mast cells express ETA receptors and release ET-1, and guinea pig lung mast cells express ETB receptors [19 ]. ET-1 has also been shown to stimulate histamine release differentially from various mast cell subtypes, reflecting mast cell heterogeneity [17 18 19 ].

There is limited information about the modulation of cytokine production by ETs. Thus, given the importance of mast cells in asthma and increased ET levels in asthmatic patients, we investigated the modulatory effect of ETs on mast cell mediator production. ET-1 and ET-3 induced a significant release of ß-hexosaminidase, a preformed mediator. It is interesting that ETs inhibited Th2-type cytokine production but stimulated the production of tumor necrosis factor (TNF) as well as interleukin (IL)-12, a Th1-type cytokine.


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MATERIALS AND METHODS
 
Cell line
The mucosal mast cell line rat tissue-cultured mast cells (RCMC)-1 has been well characterized by alcian blue-positive and safranin 0-negative staining, the presence of rat mast cell protease II, and a low histamine content [20 ]. RCMC-1 were obtained from Dr. Bosco Chan (University of Western Ontario, London, Canada) and maintained in RPMI-1640 medium containing 5% heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, 1% HEPES-buffered solution, 1% L-glutamine (Canadian Life Technologies, Burlington, ON, Canada), and 0.2% gentamicin (Novopharm, Toronto, ON, Canada). Cells were grown at 37°C in a humidified incubator with 5% CO2. Cell viability, as assessed by trypan blue-dye staining, always exceeded 95% after treatment with ETs.

Reagents
ET-1 and ET-3 were purchased from American Peptide Co. (Sunnyvale, CA). Lipopolysaccharides (LPS) from Salmonella enteritidis, ionophore A-23187, and 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide were purchased from Sigma Chemical Co. (St. Louis, MO). The ETA receptor antagonist, ABT-627 {[2R, 3R, 4S]-2-(4-methoxyphenyl)-4-(1, 3-benzodioxol-5-yl)-1-[(N, N-dibutylamino-carboxyl)methyl]pyrrolidine-3-carboxylic acid}, with an IC50 of 0.034 nM for ETA receptor and 63.3 nM for ETB receptor [21 ], and ETB receptor antagonist, A-192621.1 {[2R-(2{alpha},3ß,4{alpha})]-4-(1,3-benzodioxol-5-yl)-1-[2-[(2,6-diethylphenyl)amino]-2-oxoethyl]-2-(4-propoxyphenyl)-3-pyrrolidinecarboxylic acid}, with an IC50 of 6300 nM for ETA receptor and 4.5 nM for ETB receptor [22 ], were synthesized at Abbott Laboratories (Abbott Park, Chicago, IL).

ß-Hexosaminidase release and assay
Duplicate samples of 1 x 106 RCMC-1/ml were incubated in HEPES-buffered Tyrode solution at 37°C for 1 h before being washed with Tyrode solution and treated with different concentrations of ET-1 and ET-3 (10-11–10-8 M) for 20 min at 37°C. ß-Hexosaminidase release was determined as described previously [23 ]. Briefly, ß-hexosaminidase levels were measured in supernatants and boiled pellets by fluorometric assay using 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide and a Fluoroskan Ascent FL (Labsystems, Helsinki, Finland). ß-Hexosaminidase release is expressed as a percentage of total cellular ß-hexosaminidase content calculated by the formula [ß-hexosaminidase in supernatant/(ß-hexosaminidase in supernatant+pellet)] x 100. Specific release represents the release of ß-hexosaminidase in the presence of the stimulus minus the spontaneous release.

Cytokine release and assay
After 1 h incubation, mast cells (1x106/ml) were washed with RPMI-1640 medium and treated or not with ETA and ETB receptor antagonist for 30 min, and different concentrations (10-12–10-6 M) of ET-1 or ET-3 were added. Cell-free supernatants were collected after 18 h, and cell pellets were frozen and sonicated on ice (10 s on followed by 10 s off, three times) using the 4710 Series ultrasonic homogenizer (Cole-Palmer Instrument Co., Chicago, IL). IL-4, IL-10, IL-12, IL-13, and TNF were measured using enzyme-linked immunosorbent assay (ELISA) kits for rat (Biosource International, Camarillo, CA), whereas macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}) was measured using an ELISA that we developed [24 ]. Plates were read on a THERMOmax microplate reader (Molecular Devices, Menlo Park, CA). The sensitivity of ELISA was 2 pg/ml for IL-4, 5 pg/ml for IL-10, 5 pg/ml for IL-12, 1.5 pg/ml for IL-13, 4 pg/ml for TNF, and 7 pg/ml for MIP-1{alpha}. Preliminary experiments were done to determine the concentration of ET-1 and ET-3 (10-8 M and 10-7 M) needed to observe modulation of cytokine production.

Immunocytochemistry
Mast cells were fixed with ice-cold 4% paraformaldehyde (Sigma Chemical Co.) prepared in Dulbecco’s phosphate-buffered saline (PBS), pH 7.4. Cells were washed with cold PBS, and cytospins were performed on a charged slide. The cytospin preparations were washed with 0.05 M Tris-HCl-buffered isotonic saline (TBS). After treatment with universal blocking solution (Dako Diagnostics Canada, Mississauga, Ontario), the cytospins were incubated with anti-ETB receptor, anti-ETA receptor antibodies (Cedarlane Laboratories, Hornby, ON, Canada), or isotype control (dilution 1:40) overnight at 4°C. After washing, rabbit antisheep immunoglobulin G conjugated with horseradish peroxidase (dilution 1:200; DAKO Corp., Carpinteria, CA) was added for 30 min at 37°C and washed with TBS, and the enzyme substrate (AEC; DAKO Corp.) was added (5 min).

RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR)
Given that mRNA is expressed before the release of the protein, mast cells were treated for 2 h with ET-1 or ET-3 (10-8 M), and total RNA was isolated using the QIAGEN RNeasy mini kit (Qiagen, Mississauga, ON, Canada) from 12 x 106 RCMC-1. RNA was quantified using an Ultrospec 2000 spectrophotometer (Pharmacia Biotech, Cambridge, England). For cDNA synthesis, 1 µg total RNA was reverse-transcribed by Moloney murine leukemia virus (M-MLV) RT (Canadian Life Technologies) in a Peltier thermal cycler (PTC-200; MJ Research, Watertown, MA). PCR was performed using Thermus aquaticus (Taq) DNA polymerase (Qiagen). The primers used are listed in Table 1 . IL-12 p35 and IL-13 primers were purchased from Biosource International. Variable numbers of cycles were used to optimize the expression of PCR products for each cytokine. PCR products were separated on a 1% agarose gel (Canadian Life Technologies) containing 0.005 µg/ml ethidium bromide (Fisher Biotech, Nepean, ON, Canada). Pictures of the gels were taken using AlphaImager 2000 version 3.2 (Alpha Innotech Corp., San Leandro, CA), and the image was imported to NIH Image 1.61 analysis program for densitometric-scanning analysis. The ratio of the band density of each cytokine over a housekeeping gene, ß-actin, from the same RT was calculated.


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  		Table 1. Cytokine PCR Primers

Nitric oxide (NO) release and assay
Duplicate samples of 1 x 106 RCMC-1/ml were incubated in RPMI-1640 medium at 37°C for 1 h. After this equilibration period, mast cells were washed, and 10-8 M ET-1 or ET-3 was added for 48 h at 37°C. Levels of NO metabolites (NO2- and NO3-) were measured by gas-phase chemiluminescence [25 ] using a NO analyzer (NOA Model 280, Sievers Instruments Inc., Ionics, Boulder, CO), as described previously. The sensitivity of the assay was 1 x 10-9 M.

Statistical analysis
One-sample analysis and analysis of variance combined with Fisher’s protected least-significant difference test or Scheffe’s posteriori test for significance at P < 0.05 were used to compare treatments. Results are expressed as mean ± SE.


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RESULTS
 
Stimulation of ß-hexosaminidase release by ETs
To investigate the modulation of preformed mediator release by ETs, mast cells were treated with different concentrations of ET-1 or ET-3 for 20 min, and the level of ß-hexosaminidase was measured in cell pellets and cell-free supernatants. ET-1 (10-11 and 10-10 M) and ET-3 (10-10 M) induced a small but significant increase of ß-hexosaminidase release from RCMC-1 in a concentration-dependent manner (Fig. 1 ). The ET-induced ß-hexosaminidase release from RCMC-1 reached a peak of 14.1 ± 0.8% and 13.8 ± 1.3% following stimulation with 10-10 M of ET-1 and ET-3, respectively, compared with the spontaneous release of 6.1 ± 0.5% and 8.5 ± 0.6%. Positive control using ionophore A-23187 (1 µM) induced a specific release of ß-hexosaminidase of 10.4 ± 2.9%.



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  		Figure 1. Effect of different concentrations of ET-1 and ET-3 on RCMC-1 ß-hexosaminidase release. ET-1 (10-11 M and 10-10 M) and ET-3 (10-11 M) increased ß-hexosaminidase release significantly (*, P<0.05) compared with spontaneous release. Mean ± SE of six to eight experiments done in duplicates.

Modulation of cytokine production by ETs
In addition to the release of preformed mediators, mast cells produce proinflammatory cytokines such as TNF, as well as Th1 (IL-12) and Th2 (IL-4, IL-10, and IL-13) cytokines. To investigate the modulation of mast cell cytokine production, RCMC-1 were treated with ET-1 or ET-3 (10-8 M) for 18 h, and TNF levels were determined in cell-free supernatants and cell pellets. ET-1 and ET-3 increased TNF release significantly (74% and 70%, respectively). However, only ET-3 increased cell-associated TNF significantly (Fig. 2A ). To explore the mechanism of the modulation of TNF production by ETs further, we investigated the effects of ET-1 and ET-3 (10-8 M, 2-h treatment) on TNF-mRNA levels by RT-PCR. ET-1 (16%) and ET-3 (54%) stimulated TNF-mRNA expression significantly (Fig. 2B and 2C) .



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  		Figure 2. Modulation of TNF production by ETs. (A) ET-1 and ET-3 treatment (10-8 M, 18 h) increased TNF release (supernatant) significantly (*, P<0.05), whereas only ET-3 treatment (10-8 M, 18 h) increased cell-associated TNF (pellet) significantly compared with control (CTRL). Mean ± SE of six to nine experiments. (B) RCMC-1 were treated for 2 h with ET-1 or ET-3 (10-8 M), RNA was isolated, and RT-PCR was performed to assess TNF-mRNA expression. (C) Relative mRNA expression of TNF was quantified by densitometric analysis normalized against ß-actin using NIH Image and expressed as ratio of stimulation on control (CTRL). ET-1 and ET-3 increased TNF-mRNA expression significantly (P<0.05 and P<0.01, respectively). Mean ± SE of four to six experiments.

The modulation of one key Th1 cytokine, namely IL-12, was also investigated. Treatment of mast cells with ET-1 or ET-3 (10-7 M, 18 h) stimulated the release of IL-12 significantly (Fig. 3A ). The increase of IL-12 was similar with both ETs (39% and 41% for ET-1 and ET-3, respectively). Although both ETs increased IL-12 release, they did not modulate cell-associated IL-12 (21.4, 21.0, and 19.7 pg/106 RCMC-1 for sham, ET-1, and ET-3, respectively). To investigate the modulation of IL-12 subunits p35 and p40, we performed RT-PCR for each subunit. IL-12 p40 mRNA was expressed constitutively, whereas IL-12 p35 mRNA was barely detectable. It is interesting that ET-1 and ET-3 did not stimulate mRNA expression of IL-12 p40 but increased mRNA levels of IL-12 p35 (Fig. 3B) .



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  		Figure 3. Modulation of IL-12 production by ETs. (A) ET-1 and ET-3 treatment (10-7 M, 18 h) increased IL-12 release significantly (*, P<0.05) compared with control (CTRL). Mean ± SE of three experiments. (B) RCMC-1 were treated for 2 h with ET-1 or ET-3 (10-8 M), RNA was isolated, and RT-PCR was performed to assess IL-12 p40 and p35 mRNA expression. Densitometric analysis did not show significant modulation of mRNA IL-12 p40 expression by ETs (six to seven experiments), but increased expression of IL-12 p35 was observed.

RCMC-1 cell pellets contained low amounts of Th2 cytokines, IL-4, IL-10, and IL-13 (2.7, <5, and 6.9 pg/106 cells, respectively), and the levels of these cytokines were not modulated significantly by ET-1 (1.7, <5, and 5.8 pg/106 cells, respectively) or ET-3 (1.8, <5, and 6.6 pg/106 cells, respectively) at 10-8 M for 18 h. Furthermore, no IL-10 or IL-4 was detected in cell-free supernatants of unstimulated cells, whereas the IL-13 level reached 22.5 pg/106 cells. Similarly, neither ET-1 nor ET-3 modulated the release of these Th2 cytokines (unpublished results). Given the limited basal production of these cytokines by RCMC-1, the modulation of IL-4, IL-10, and IL-13 mRNA levels was investigated by RT-PCR (Fig. 4A ). It is interesting that ET-1 and ET-3 inhibited (25–30%) mRNA expression of IL-4, IL-10, and IL-13 significantly (Fig. 4B) .



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  		Figure 4. Modulation of Th2-type cytokine mRNA expression by ETs. (A) RCMC-1 were treated with ET-1 or ET-3 (10-8 M) for 2 h, RNA was isolated, and RT-PCR was performed to assess mRNA expression. (B) Relative mRNA expression of Th2 cytokines was quantified by densitometric analysis normalized against ß-actin using NIH Image and expressed as ratio of stimulation on control (CTRL). ET-1 and ET-3 inhibited IL-4, IL-10, and IL-13 mRNA expression significantly (P<0.05). Mean ± SE of five to seven experiments.

Modulation of MIP-1{alpha} production by ETs
To investigate the modulatory effect of ETs on chemokine production, RCMC-1 were treated with ET-1 or ET-3 for 18 h, and MIP-1{alpha} content was determined in cell pellets and cell-free supernatants. MIP-1{alpha} was undetectable in RCMC-1 cell pellets treated or not with ETs (unpublished results). Furthermore, both ETs (10-8–10-6 M) failed to induce the release of MIP-1{alpha} following 18 h treatment. However, RCMC-1 were able to release MIP-1{alpha} when stimulated with LPS (10 µg/ml) for 18 h (73.7±4.6 pg/106 cells compared with 0 pg/106 cells in control; n=3; P<0.01). Given the results obtained with the modulation of mRNA expression of Th2-type cytokines, MIP-1{alpha} mRNA expression was investigated in mast cells treated with ETs for 2 h. ET-1 and ET-3 failed to modulate MIP-1{alpha} mRNA expression significantly, whereas LPS increased it (Fig. 5A ).



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  		Figure 5. Modulation of MIP-1{alpha} and iNOS mRNA expression. RCMC-1 were treated with ET-1, ET-3 (10-8 M), or LPS (10 µg/ml) for 2 h, RNA was isolated, and RT-PCR was performed to assess MIP-1{alpha} mRNA expression. (A) LPS but not ET-1 or ET-3 stimulated MIP-1{alpha} mRNA expression in RCMC-1 compared with control (CTRL). (B) iNOS mRNA expression was not modulated by ET-1 and ET-3.

Modulation of NO production by ETs
RCMC-1 were treated with ETs (10-8 M) for 48 h, and NO metabolites (NO2- and NO3-) were measured in cell-free supernatants. Under these conditions, ET-1 and ET-3 did not stimulate NO release (control, 0.82±0.2 µM/106 cells; ET-1, 0.87±0.17 µM/106 cells; and ET-3, 0.82±0.22 µM/106 cells). Therefore, the modulation of mRNA expression of the inducible form of NO synthase (iNOS) was investigated. ET-1 and ET-3 treatments (10-8 M, 2 h) failed to induce iNOS mRNA expression (Fig. 5B) .

ET receptors on RCMC-1
To investigate which ET receptors were involved in the stimulation of TNF release form RCMC-1, cells were pretreated for 30 min with a selective ETA receptor antagonist, ABT-627, or ETB receptor antagonist, A-192621.1, before being stimulated with ETs (10-8 M). ET receptor antagonists did not modulate the spontaneous release of TNF (Fig. 6 ). However, ET-1- and ET-3-stimulated TNF release was abrogated by the presence of ETB receptor antagonist (10-7 M). ETA receptor antagonist did not modulate the release of ET-stimulated TNF release significantly.



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  		Figure 6. Modulation of TNF release by ET receptor antagonists. RCMC-1 were treated with 10-7 M ABT-627 (ETA-selective) or A-192621 (ETB-selective) for 30 min before the addition of ET-1 or ET-3. ET-1 and ET-3 treatments (10-8 M, 18 h) increased TNF release significantly (*, P<0.05). ETB receptor antagonist inhibited ET-induced TNF increase significantly ({ddagger}, P<0.05). Mean ± SE of nine experiments.

To further identify ET receptors on rat mucosal mast cells (RCMC-1), immunocytochemical staining was performed using specific antibodies against ETA and ETB receptors. It is interesting that a specific staining was observed with anti-ETA and anti-ETB antibodies (Fig. 7B and 7C ). The substitution of the first antibody with an isotype control eliminated the immunostaining of the positive cells, demonstrating the specificity of the analysis (Fig. 7A) . These results indicate that ETA and ETB receptors are present on RCMC-1.



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  		Figure 7. Representative immunocytochemical staining for ETA and ETB receptor subtypes from rat mucosal mast cells, RCMC-1. (A) Absence of immunostaining in the isotype control experiments. (B) Positive immunoreactivity with mouse anti-rat ETA receptors in RCMC-1. (C) Positive immunoreactivity with mouse anti-rat ETB receptors in RCMC-1. Original magnification, x40.


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DISCUSSION
 
Given the plethora of biological actions induced by ETs, their potential role in asthma, and the wide distribution of mast cells, the modulation of mast cell mediator production by ETs is of great interest. Furthermore, the observation that mast cells produce ETs and express ET receptors suggests that ETs may modulate mast cell function in an autocrine manner [16 17 18 ]. Although the roles of ETs in the cardiovascular system have been investigated more extensively [26 ], the modulation of the immune response by ET deserves further investigation. In this study, we have shown that ET-1 and ET-3 modulate mediator production in a rat mucosal mast cell line, RCMC-1. ET-1 and ET-3 increased RCMC-1 ß-hexosaminidase release (8.0% and 5.3%) at 10-10 M, but they did not have any significant effect at a higher concentration. This may be caused by receptor desensitization as demonstrated previously [27 ]. The increase of ß-hexosaminidase was similar to ET-1-stimulated histamine release (4%) from murine bone marrow-derived mast cells [17 ], suggesting that ETs are not potent inducers of mucosal mast cell degranulation in rodents. However, ionophore A-23187, which is a potent mast cell degranulator, induced only 10.4% release of ß-hexosaminidase, suggesting that maximal stimulation of these cells to release ß-hexosaminidase is limited and that ET-1 is nevertheless as potent as ionophore.

The modulation of ET-1 production by various cytokines is well documented [28 ]. However, there is limited information about the modulation of cytokine production by ETs. ET-1 has been shown to modulate IL-6 production in endothelial cells [29 ], to induce TNF, IL-1, IL-6, and IL-8 production in monocytes, and to increase TNF release from human alveolar macrophages isolated from bronchoalveolar lavage of patients with stable asthma [30 , 31 ] and cultured macrophages [32 ]. It is interesting that TNF, like several other cytokines, has been shown to stimulate ET-1 secretion from cultured bovine airway smooth-muscle cells and animal and human airway epithelial cells [33 , 34 ], suggesting a possible regulatory loop between ET-1, TNF, and other cytokines. Our results showed an increase in ET-induced TNF production as demonstrated in human monocytes and alveolar macrophages [30 31 32 ]. ET-1 and ET-3 also stimulated TNF-mRNA expression in RCMC-1. However, ET-1 did not modulate cell-associated TNF significantly in contrast to ET-3. Thus, ET-1 stimulates mast cells to release TNF but not to store it inside the cells, whereas ET-3 induces the release and storage of TNF. These results may also reflect a difference in the kinetics of TNF release after ET-1 and ET-3 treatment, as observed at mRNA levels. It is interesting that there is some evidence suggesting an important role of TNF in the development of the Th1 response [35 ]. Thus, ETs may favor a Th1-type response in stimulating mast cell TNF release.

IL-12 is a 70 kDa heterodimeric cytokine, composed of 35 kDa and 40 kDa subunits linked by disulfide bonds. Although each subunit is controlled independently, the expression of p35 determines the level of active IL-12 protein [36 ]. In contrast, the p40 subunit exists as a dimer, which can bind to IL-12 receptors, and act as an IL-12 antagonist in vivo and in vitro [37 , 38 ]. Although ET-1 and ET-3 treatment increased the release of IL-12 and mRNA levels of IL-12 p35, they did not modulate mRNA expression of the p40 subunit. However, the stimulation of IL-12 p40 mRNA expression may need more than 2 h treatment with ETs (time used in the study of mRNA expression), explaining the increase in IL-12 release after 18 h treatment without modulation of IL-12 p40 mRNA levels. Thus, with the inhibitory effects of ETs on the production of Th2 cytokines and the stimulatory effect of Th1 cytokine production, ETs may participate in the equilibration of Th1/Th2 cytokines in asthma.

RCMC-1 did not have preformed MIP-1{alpha}, but they release it after LPS stimulation. ETs failed to modulate MIP-1{alpha} production at the mRNA and protein levels, suggesting that ETs may not participate in the recruitment of inflammatory cells, at least via the production of MIP-1{alpha}. However, other chemokines, such as regulated on activation, normal T expressed and secreted (RANTES) and eotaxin, could be modulated by ET treatment. In addition to chemokine production, mast cells have also been shown to produce NO [39 ]. However, ETs did not modulate the release of NO or the expression of iNOS mRNA in rat mucosal mast cells. Similar data have demonstrated the lack of modulation of iNOS mRNA expression by ET-1 in human bronchial epithelial cells [40 ].

Our data demonstrated similar effects of ET-1 and ET-3 on mast cell mediator production, suggesting the activation of ETB receptor. Furthermore, ETB receptor antagonist abrogated ET-1, and ET-3 induced TNF release, suggesting the presence of ETB receptor on RCMC-1. It is interesting that the immunocytochemistry analysis demonstrated the presence of ETA and ETB receptors on RCMC-1. Rat intestinal mucosal mast cells have also been shown to express both ET receptors [41 ]. Our results suggest that ET-stimulated TNF release is mediated by ETB. Given that ETs also increase IL-12 release, it is tempting to speculate that ET-related induction of cytokines may be mediated by ETB, whereas ET-related inhibition of cytokines, such as the effect of ET-1 and ET-3 on Th2 (IL-4, IL-10, IL-13) cytokines, may be mediated by ETA receptors. However, the ETA receptor has been shown to be involved in ET stimulation of TNF release by cultured macrophages [32 ]. Thus, the role of this receptor subtype on mast cell functions needs further investigation.

Th2 cytokines play a major role in the pathophysiology of allergic diseases such as asthma. Intriguingly, our study suggests that the increased level of ETs observed in asthma may contribute to the production of Th1-type cytokines and the inhibition Th2-type cytokines by mast cells. Thus, in addition to their bronchoconstrictor effects, ETs may modulate mast cell functions to counter-balance high levels of Th2 cytokines present in asthma. However, given that rat mast cell lines were used in this study, further investigations using human mast cells are needed to better understand the role of ETs in asthma pathogenesis.


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ACKNOWLEDGEMENTS
 
This work was supported by "La Chaire de Pneumologie de la Fondation J. D. Bégin" and "Les Fonds pour la Recherche en Santé du Québec (FRSQ)". E. Y. B. is a senior FRSQ scholar, and B. B. is a junior II FRSQ scholar. The authors thank J. Sirois, G. Ménard, and S. Molez for their expert technical assistance.

Received August 28, 2001; revised October 26, 2001; accepted November 3, 2001.


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