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Originally published online as doi:10.1189/jlb.0906548 on February 8, 2007

Published online before print February 8, 2007
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(Journal of Leukocyte Biology. 2007;81:1245-1251.)
© 2007 by Society for Leukocyte Biology

Modulation of eosinophil activation in vitro by a nicotinic receptor agonist

Marie-Renée Blanchet, Anick Langlois, Evelyne Israël-Assayag, Marie-Josée Beaulieu, Claudine Ferland, Michel Laviolette and Yvon Cormier1

Centre de Recherche, Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie de l’Université Laval, Ste-Foy, Québec, Canada

1 Correspondence: Centre de Pneumologie, Hôpital Laval, 2725 Chemin Ste-Foy, Ste-Foy, Québec, Canada, G1V 4G5. E-mail: yvon.cormier{at}med.ulaval.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nicotinic receptor agonists decreased the infiltration of eosinophils into the lung and airways in a mouse model of asthma. To better understand the mechanisms implicated in this anti-inflammatory phenomenon, the expression of nicotinic acetylcholine receptors (nAChRs) and the effect of dimethylphenylpiperazinium (DMPP), a nonselective nAChR agonist, on human blood eosinophils were studied. The expression of {alpha}-3, -4, and -7 nAChR subunits on human blood eosinophils was measured by cell ELISA and immunocytochemistry. mRNA expression for all three subunits was evaluated by quantitative RT-PCR. The effect of DMPP on leukotriene C4 (LTC4) and matrix metalloproteinase-9 (MMP-9) production, eosinophil migration, and intracellular calcium mobilization was measured. The results show that the {alpha}-3, -4, and -7 nAChR subunits and mRNAs are expressed by blood eosinophils. In vitro treatment of these cells with various concentrations of DMPP reduced platelet-activating factor (PAF)-induced LTC4 production significantly. DMPP (160 µM) decreased eotaxin, and 5-oxo-6,8,11,14-eicosatetranoic acid induced eosinophil migration through Matrigel by 40.9% and 55.5%, respectively. This effect was reversed by the nAChR antagonist mecamylamine. In addition, DMPP reduced MMP-9 release and the inositol 1,4,5-triphosphate-dependent intracellular calcium increase provoked by PAF. Taken together, these results indicate that functional nAChRs are expressed on eosinophils and that nAChR agonists down-regulate eosinophil function in vitro. These anti-inflammatory effects could be of interest in the treatment of allergic asthma.

Key Words: chemotaxis • dimethylphenylpiperazinium • leukotriene • intracellular calcium


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Infiltration of eosinophils into the lung tissue is a major feature of asthma [1 ]. Eosinophils migrate to the inflammatory site in response to chemotactic mediators such as eotaxin and 5-oxo-6,8,11,14-eicosatetranoic acid (5-oxo-ETE) [2 , 3 ]. Once they have reached the bronchial mucosa, activated eosinophils are a main source of leukotrienes (LTs), which can promote bronchial smooth muscle constriction, eosinophil recruitment, mucus secretion, and airway hyper-responsiveness [4 ]. Eosinophils are also a source of matrix metalloprotease-9 (MMP-9), a gelatinase involved in the pathogenesis of asthma [5 ]. MMP-9 is in part responsible for the degradation of extracellular matrix (ECM) components [6 ]. Platelet-activating factor (PAF)-induced migration of eosinophils is regulated by inositol 1,4,5-triphosphate (IP3)-dependent calcium increase [7 ] via the stimulation of the G protein-coupled PAF receptor [8 ] and by the release of MMPs such as MMP-9 [9 ]. Similarly, 5-oxo-ETE and eotaxin-induced migration are also dependent on cytosolic calcium increase and protease activation [10 , 11 ].

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels made up of five subunits. These receptors are widely expressed on lymphocytes [12 13 14 ], alveolar macrophages [12 ], airway smooth muscle cells [15 ], epithelial cells [16 ], and fibroblasts [17 ]. Stimulation of the {alpha}-7 nAChR subunit is responsible for the inhibition of TNF release by macrophages via the cholinergic, anti-inflammatory pathway [18 ]. Nicotinic agonists, such as nicotine or dimethylphenylpiperazinium (DMPP), have anti-inflammatory effects in mouse models of hypersensitivity pneumonitis [19 ], asthma [20 ], airway inflammation [21 , 22 ], and Type I diabetes [23 ]. In addition, {alpha}-5 nAChR subunit-deficient mice showed an increased severity in a mouse model of colitis [24 ].

In vitro, nicotine down-regulates the alveolar macrophage release of TNF and IL-1ß [25 ], reduces lymphocyte proliferation, and blocks these cells in the G0/G1 state [26 ]. Moreover, previous studies suggested that the suppression of T cell responsiveness by nicotine results from the depletion of IP3-sensitive intracellular calcium stores [27 ]. DMPP, another nicotinic agonist, reduces the release of TNF and IL-1ß from isolated spleen cells [28 ] and TNF and IL-6, by alveolar macrophages [29 ]. We have also reported that the anti-inflammatory effect of DMPP is mediated by PI-3K activation and intracellular calcium store depletion [30 ].

It is interesting that neither nAChR expression on eosinophils nor the effects of nicotinic agonists on eosinophil functions have been reported. We have shown previously that treatment with DMPP in a mouse model of asthma reduced eosinophil recruitment into the lung [20 ]. However, the direct effect of nicotinic agonists on eosinophil functions is still unknown. In this study, we demonstrate the presence of {alpha}-3, -4, and -7 nAChR subunits on purified blood eosinophils and the effect of the nicotinic agonist DMPP on LTC4 release, eotaxin, 5-oxo-ETE-induced eosinophil migration, MMP-9 release, and intracellular calcium mobilization in vitro.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subject selection
Mild asthmatics (nonsmokers) meeting the American Thoracic Society criteria for asthma [31 ] and taking ß2 agonists on demand were recruited as blood donors. Approval from the local Ethics Committee was obtained, and subjects signed informed consent forms. All subjects underwent blood sampling between 8:00 a.m. and 10:00 a.m.

Isolation of blood eosinophils
Eosinophils were isolated as described previously [2 , 3 ]. Briefly, blood was centrifuged to remove platelet-rich plasma, and the resulting pellet was sedimented for 30 min on a 6% Dextran solution. Leukocytes were resuspended and centrifuged on Ficoll-Paque. The granulocyte layer was resuspended, and RBC were lysed with distilled water. Eosinophils were purified from neutrophils by negative selection using bead-conjugated anti-CD16 (Fc{gamma}RIII) mAb and a magnetic cell sorter and counted with a hemacytometer. The purity of the eosinophil preparation was always >98%. Purified eosinophils were incubated with recombinant human IL-5 (10 ng/ml; PeproTech, Rocky Hill, NJ, USA) in RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin, unless otherwise stated. Eosinophil survival after the isolation procedures was assessed by trypan blue exclusion.

Cell ELISA for nAChR subunit expression
Cell ELISA was performed as described previously [32 ] with minor modifications. Eosinophils (1x105 per well) were adsorbed onto 96-well plates by evaporation overnight at room temperature. Cells were then fixed with methanol at –20°C for 20 min. Endogenous peroxidase and pseudoperoxidase activity were blocked with Dako peroxidase-blocking reagent (DakoCytomation, Denmark) at room temperature for 10 min. The wells were blocked with 1% BSA at 37°C for 60 min and incubated at 37°C for 60 min with 5 µg/ml rabbit anti-{alpha}-3, -4, and -7 human nAChR (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody or rabbit IgG (Alpha Diagnostic International, San Antonio, TX, USA) as isotype control in PBS 0.1% BSA + 0.05% Tween 20. Binding was revealed with peroxidase-conjugated goat antirabbit IgG (H+L) (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) in PBS + 0.05% Tween 20 at 37°C for 60 min, followed by the addition of a substrate solution (R&D Systems Inc., Minneapolis, MN, USA). Reaction was stopped after 10 min with H2SO4 2N solution, and OD was read at 450 nm with correction at 550 nm (Thermoplax microplate reader, Molecular Devices Corp., Sunnyvale, CA, USA).

Immunocytochemistry for nAChR subunit expression
{alpha}-3, -4, and -7 nAChR subunit expression was also assessed by immunocytochemistry. Cytospin preparations were made with eosinophils (75,000 per slide) and were fixed in 4% paraformaldehyde. Endogenous peroxidase was quenched by incubating the slides with 0.5% hydrogen peroxide in TBS for 30 min. Cells were blocked with blocking solution (DakoCytomation) for 30 min at room temperature and incubated overnight at 4°C with the primary antibody (40 µg/ml) in diluting buffer (DakoCytomation) of rabbit polyclonal anti-{alpha}-3, -4, or -7 human nAChR (Santa Cruz Biotechnology) or isotype control (rabbit IgG; Southern Biotech, Birmingham, AL, USA). Preparations were washed with TBS and incubated for 30 min with EnVision system HRP-labeled polymer antirabbit (DakoCytomation). Nicotinic acetylcholine receptor expression was revealed using AEC substrate chromogen (DakoCytomation). Sections were counterstained with hematoxylin and mounted.

RT-PCR for nAChR mRNA expression
Total RNA from purified eosinophils was extracted with TRIzol reagent, according to the manufacturer’s instructions (Invitrogen Canada, Burlington, Ontario). cDNA synthesis was performed using the IscriptTM cDNA synthesis kit, according to the manufacturer’s recommendations. PCR amplification was performed using the IQTM SYBR Green supermix (Bio-Rad Laboratories, Mississauga, Ontario, Canada) containing iTaq DNA polymerase, deoxy-unspecified nucleoside 5'-triphosphate mix, buffer, MgCl2, and fluorescent dye. cDNAs were amplified with QuantiTect human nicotinic receptor {alpha}-3, -4, and -7 and GAPDH primers (Qiagen, Mississauga, Ontario, Canada). RT-PCR reactions were carried out on DNA engine Opticon2 (MJ Research, Watertown, MA, USA) for 40 cycles with the following settings: denaturation at 94°C for 20 s (3 min for the first cycle), annealing at 55°C for 40 s, and extension at 72°C for 40 s. Fluorescence was measured at the end of every cycle to allow quantification of cDNA. After amplification, a melting curve was obtained by slowly heating from 30°C to 95°C with fluorescence detection every 1°C following normal cycle. Agarose gel electrophoresis analysis was used to assess the specificity of the amplification products.

LTC4 assay
Eosinophils were incubated in complete RPMI with 10 ng/ml IL-5 for 18 h, with or without increasing doses of DMPP. Cells (1x106) were stimulated with 1 µM PAF in HBSS-CaCl2 for 10 min before the addition of 0.1 µM C5a for 10 more min. The reactions were stopped by putting the cells on ice. LTC4 was measured in the supernatants by quantitative immunoassay according to the manufacturer’s recommendations (Cayman Chemical, Ann Arbor, MI, USA).

Migration assay
To evaluate the effect of DMPP on eotaxin and 5-oxo-ETE-induced migration, eosinophils (1x106 in RPMI, supplemented with 10% FBS+0.2% penicillin/streptomycin+10 ng/ml IL-5) were preincubated with or without increasing doses of DMPP for 30 min and then placed in the upper chamber of 24-well Biocoat Matrigel invasion chambers (BD Biosciences, San Jose, CA, USA). Eotaxin (10 nM) or 5-oxo-ETE (1 µM) was added in the lower chamber as a chemoattractant. In some experiments, the nicotinic antagonist mecamylamine (15 µM) was added 15 min prior to DMPP to assess the specificity of the reaction to the nAChR. Chambers were incubated at 37°C + 5% CO2 for 18 h. At the end of the incubation period, cells from the upper and lower chambers were removed by aspiration and counted on a hemacytometer. For each condition, the percentage of migration was calculated as the number of cells in the lower chamber of the Matrigel invasion chamber divided by the number of cells in the lower chamber of a control invasion chamber without the Matrigel membrane and multiplied by 100.

Immunoprecipitation of MMP-9 and Western blot
Cells (3x106) were preincubated with or without DMPP (160 µM) for 30 min and stimulated with 5-oxo-ETE (1 µM) for 18 h. Supernatants were collected, and MMP-9 from supernatants was immunoprecipitated with Sepharose A gel beads coupled to a mouse antihuman MMP-9 (R&D Systems Inc.). Immunoprecipitates were run on a 10% SDS-PAGE. Proteins were transferred by Western blot on a polyvinylidene difluoride membrane, which was incubated with the same mouse antihuman MMP9 antibody used for the immunoprecipitate, followed by a goat antimouse coupled to HRP (Cederlane Laboratories Ltd.). Revelation was made using ECL staining (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and the intensity of the bands was quantified (Scion Image, National Institutes of Health, Bethesda, MD, USA).

Intracellular calcium studies
Eosinophils (0.25x106) were plated in a 2.5-cm petri dish coated with 25 µg/ml Matrigel (Becton Dickinson, Bedford, MA, USA) in complete RPMI + IL-5 (10 ng/ml) and incubated with 2.5 µM Fura-2-AM for 30 min at 37°C. Cells were rinsed to remove extracellular Fura-2. Cells were pretreated or not for 24 h with 160 µM DMPP and stimulated with 10 µM PAF in calcium buffer to induce intracellular calcium mobilization. Changes in intracellular calcium were followed by Fura-2 fluorescence using the Imagemaster system (Photon Technology International, Monmouth Junction, NJ, USA) coupled to a Leica DM IRB fluorescence microscope (40x; Leica Canada Inc., St-Laurent, Quebec). Excitation wavelengths were 353 nm and 374 nm, and the emission wavelength was 510 nm. Emitted fluorescence at 510 nm was recorded every 20 s. The ratio between the fluorescence provoked by the 353 nm and the 374 nm excitation wavelengths was calculated and used as an intracellular calcium concentration indicator.

Statistical analysis
Statistical analyses for the LTC4 and migration studies were done using an ANOVA table followed by a Fisher protected least-significant difference post-hoc test. For the MMP-9 densitometry, a paired t test was used to define statistical significance (P<0.05). An unpaired t test was used in the calcium studies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Eosinophils express {alpha}-3, -4, and -7 nAChR subunits and mRNA
Expression of {alpha}-3, -4, and -7 nAChR subunits on purified blood eosinophils was verified by cell ELISA using subunit-specific antibodies and by immunohistochemistry. Results presented in Figure 1A show that eosinophils are positive for all three nAChR subunits. We did not see any significant change in subunit expression when cells were treated with 160 µM DMPP for 24 h (data not shown). The cell ELISA results were confirmed further by positive immunochemistry staining of eosinophil cytospin preparations (Fig. 1B) . RT-PCR was used to verify mRNA production, and results are presented in Figure 1C . Blood eosinophils were positive for {alpha}-3, -4, and -7 nAChR mRNA. Moreover, under our amplification conditions, subunit PCR products were detectable after 31, 33, and 24 cycles, respectively.


Figure 1
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  		Figure 1. Human blood eosinophils express nAChRs. (A) Cell ELISA for {alpha}-3, -4, and -7 nAChR subunits on blood eosinophils. Results are presented as mean 490 nm OD intensity x 1000 – control Ig OD value. Each column represents the mean ± SEM for purified blood eosinophils from seven subjects. (B) Immunohistochemistry for expression of {alpha}-3, -4, and -7 nAChR subunits on blood eosinophils. Arrows indicate positive cells. (C) RT-PCR products for {alpha}-3, -4, and -7 nAChR subunit mRNA detection (this is a representative result of four different experiments).

 
DMPP reduces LTC4 release by eosinophils
The stimulation of cells with PAF-C5a (Fig. 2 ) induced the release of LTC4 by eosinophils. Pretreatment with 80, 160, or 320 µM DMPP decreased LTC4 production to 33.98% ± 7.37, 35.25% ± 5.50, and 27.84% ±17.60 of controls, respectively (n=4–8; P<0.001). Eosinophil survival (as assessed by trypan blue exclusion) was not affected by the 24-h incubation with 160 µM DMPP (data not shown).


Figure 2
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  		Figure 2. Effect of increasing DMPP doses on LTC4 production. Results expressed as a percentage of LTC4 release. Release (100%) was attributed to PAF stimulated/untreated cells. *, P < 0.001.

 
DMPP decreased eotaxin and 5-oxo-ETE-induced eosinophil migration
To determine the effect of DMPP on eosinophil migration, different concentrations of this nicotinic agonist were used (40–320 µM; Fig. 3 ). The dose-response experiments show that the maximal effect of DMPP on eotaxin-induced eosinophil migration was obtained with 160 µM, a dose that inhibited the migration by 40.69% ± 6.43 (n=11; P<0.0001; Fig. 3A ). This optimal dose was thereafter used in the subsequent experiments. Eosinophil viability at the end of the assay (assessed by trypan blue exclusion) was greater than 95%. To test if this inhibitory effect were specific to nAChR activation, 15 µM mecamylamine (a nonselective nicotinic antagonist) was added into the eotaxin-induced migration chambers (Fig. 3B) . In these experiments, 160 µM DMPP inhibited migration by 33.8% ± 5.8, whereas adding mecamylamine abrogated this inhibition to 0.2% ± 1.33 (n=4; P=0.004). Finally, a similar inhibitory effect by DMPP of 55.57 ± 14.4% (n=5; P=0.0009) was observed on 5-oxo-ETE-induced eosinophil migration (Fig. 3C) .


Figure 3
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  		Figure 3. Effect of DMPP on eosinophil transmigration. Results of the migration studies are presented as a percent inhibition of migration (compared with the nontreated cells). (A) Percent inhibition of migration by increasing doses of DMPP in eotaxin-induced migration (n=11); *, P < 0.05. (B). Reversal of the inhibitory effect of DMPP by addition of mecamylamine (MECA), a nicotinic receptor antagonist. (C) Percent migration induced by 5-oxo-ETE in DMPP-treated (160 µM) and nontreated cells (n=5).

 
DMPP reduces MMP9 release by eosinophils
Immunoprecipitation techniques were used to concentrate supernatant MMP-9, which was undetectable by ELISA or Western blots. At 18 h, 5-oxo-ETE induced the release of MMP-9 by eosinophils (Fig. 4A ). DMPP reduced the 5-oxo-ETE-induced release of MMP-9 in supernatant. As the antibody used for the immunoprecipitation was the same as for the Western blot revelation, we used the 50-kD band, which corresponds to the antibody heavy chain as the internal control to make sure that the total quantity of protein loaded on the SDS-PAGE was the same in each condition.


Figure 4
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  		Figure 4. Effect of DMPP on the release of MMP-9 by 5-oxo-ETE-stimulated eosinophils. (A) Immunoprecipitation was used to concentrate supernatant MMP-9 from 5-oxo-ETE-stimulated eosinophils. (B) Densitometry analysis of the bands (n= 4; P=0.01).

 
Density of the bands was quantified, and a ratio between the density of the MMP-9 and the density of the Ig band was calculated. Results are presented in Figure 4B and are expressed as fold-increase compared with the ratio of the nonstimulated cell bands (being onefold). Densitometry confirmed that the treatment with 160 µM DMPP provoked a significant decrease on the release of MMP-9 by eosinophils (n=4; P=0.01).

DMPP reduced intracellular, PAF-induced calcium mobilization
Intracellular calcium mobilization in eosinophils was induced by PAF. The treatment of cells with 160 µM DMPP for 24 h delayed the increase in intracellular calcium by 253.6 ± 46.5 s (n=12; P=0.0002). The mean peak fluorescence ratio in PAF stimulated:nontreated cells was 0.912 ± 0.09 and was reduced to 0.684 ± 0.05 in PAF-stimulated:DMPP-treated cells (n=12; P=0.03; Fig. 5 ).


Figure 5
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  		Figure 5. Effect of DMPP on intracellular calcium mobilization provoked by PAF. Single cell traces of intracellular calcium mobilization provoked by the addition of PAF in 160 µM DMPP-treated or untreated cells. This is a representative result of four different experiments each consisting of recording the calcium mobilization in three different cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study was conducted to verify if nAChRs were expressed in eosinophils and if activation of these receptors could down-regulate eosinophil functions in vitro. The demonstration that eosinophils express {alpha}-3, -4, and -7 nAChR subunits and mRNA, as do other immune cells, suggests that the effects of DMPP observed in vivo in the mouse model of asthma [20 ] occurred at least in part via the direct effect of these agonists on eosinophils, in addition to the modulation of cytokine and or chemokine release by other cells such as alveolar macrophages [28 , 29 ]. It was not surprising that DMPP, which is known to present a greater affinity for {alpha}-4 [33 ] and {alpha}-7 subunits [34 ], affected eosinophil functions. However, further studies will be needed to understand better which specific receptor subunit is responsible for the modulation of different eosinophil functions and whether addition of DMPP affects nAChR expression on eosinophils.

Eosinophil migration and LTC4 release are implicated in the development of allergic asthma [1 , 2 ]. Blockade of these two processes by DMPP, as reported here, clarifies the mechanisms underlying our previously published results indicating that DMPP reduces the number of eosinophils in lung tissue and bronchoalveolar lavage and decreases airway hyper-responsiveness [20 ]. Mecamylamine reversed the inhibitory activity of DMPP on eosinophil migration, suggesting that this effect is specific to nAChR activation. This nAChR antagonist was preferred to {alpha}-bungarotoxin ({alpha}-7 nAChR-specific antagonist) or any other specific antagonist, as DMPP is a general agonist and could, in theory, activate any nAChR subunit present on eosinophils. An interesting fact is that eotaxin and 5-oxo-ETE-induced migration were inhibited by DMPP, reinforcing the idea that this molecule has a general anti-inflammatory effect.

It has been reported that 5-oxo-ETE predominantly activates the MMP-9 pathway [2 ], a gelatinase involved in the degradation of ECM. Moreover, we have shown that within 1 h of stimulation, 5-oxo-ETE provokes a significant release of MMP-9 [35 ]. When cells were treated with DMPP and stimulated with 5-oxo-ETE within that timeframe (1 h), DMPP did not affect MMP-9 activity (data not shown). Absence of serum during long incubation periods affected eosinophil viability, limiting zymography assays to short-term periods. Consequently, immunoprecipitation was performed on cell-free supernatants to assess for the presence of MMP-9 after an incubation of 18 h. Indeed, a reduction of MMP-9 release was found in the supernatant of DMPP-treated/5-oxo-ETE-stimulated eosinophils. The small amounts of MMP-9 released by eosinophils are consistent with previous reports [36 ].

The PAF receptor is a G protein-coupled receptor, which provokes an initial IP3-dependent, quick rise in intracellular calcium [37 ], followed by a reduced and more sustained response (corresponding to the calcium-induced calcium release) [38 ]. The treatment of eosinophils with DMPP delayed and reduced the IP3-dependent response. This could be a result of a lack of IP3 production by phospholipase C (PLC) or endoplasmic reticulum (ER) calcium store depletion. These results support those of previous studies indicating that nicotinic agonists depleted the ER calcium stores in lymphocytes [27 ] and monocytes [30 ]. Intracellular calcium mobilization is involved in LTC4 synthesis and eosinophil migration [4 , 11 ]. LTC4 is generated through the action of cytosolic PLA2, in concert with 5-lipoxygenase, two calcium-dependent enzymes (reviewed in ref. [39 ]). It therefore seems likely that blockade of intracellular calcium mobilization is a common mechanism for the inhibition in LTC4 release and cell migration.

The exact intracellular signaling provoked by nAChR activation on eosinophils is still poorly understood. The results presented here confirm that the nicotinic agonist DMPP has a potential down-regulating effect on eosinophil functions. These results also support the inhibitory effect of DMPP in a mouse model of asthma [20 ] and the potential anti-inflammatory role of nicotinic agonists. However, further studies will be needed to better define the intracellular mechanisms underlying these anti-inflammatory effects.

The results presented in this study confirm that functional nAChRs are expressed on eosinophils and that the nicotinic agonist DMPP inhibits LTC4 production, eosinophil migration, MMP-9 production, as well as intracellular calcium mobilization. The overall inhibitory effect of DMPP on eosinophils suggests that this molecule or other nicotinic agonists could potentially be used in the treatment of allergic asthma.


   ACKNOWLEDGEMENTS
 
This work was funded by the Institut de Recherche Robert-Sauvé en Santé et Sécurité au Travail and by the Canadian Institute of Health Research. M-R. B. and E. I-A. are coauthors of Patent #PCT/CA02/00412 (March 2002), which is owned by Laval University. Y. C. is the principal author of Patent #PCT/CA02/00412. The authors thank Marc Veillette for his precious counseling and help with the mRNA detection.

Received September 5, 2006; revised January 8, 2007; accepted January 10, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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