AJP - Lung Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 291: L757-L763, 2006. First published June 16, 2006; doi:10.1152/ajplung.00409.2005
1040-0605/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/4/L757    most recent
00409.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blanchet, M.-R.
Right arrow Articles by Cormier, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blanchet, M.-R.
Right arrow Articles by Cormier, Y.

Dimethyphenylpiperazinium, a nicotinic receptor agonist, downregulates inflammation in monocytes/macrophages through PI3K and PLC chronic activation

Marie-Renée Blanchet, Evelyne Israël-Assayag, Pascal Daleau, Marie-Josée Beaulieu, and Yvon Cormier

Hopital Laval, Centre de Recherche, Institut Universitaire de Cardiologie et de Pneumologie de l'Université Laval, Ste-Foy, Quebec, Canada

Submitted 22 September 2005 ; accepted in final form 6 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 
Activation of nicotinic acetylcholine receptors (nAChRs) on inflammatory cells induces anti-inflammatory effects. The intracellular mechanisms that regulate this effect are still poorly understood. In neuronal cells, nAChRs are associated with phosphatidylinositol 3-kinase (PI3K). This enzyme, which can activate phospholipase C (PLC), is also present in monocytes. The aim of this study was to assess the role of these proteins in the signaling pathways involved in the anti-inflammatory effect of dimethylphenylpiperazinium (DMPP), a synthetic nAChR agonist, on monocytes and macrophages. The results indicate that PI3K is associated with {alpha}3, -4, and -5 nAChR subunits in monocytes. The PI3K inhibitors wortmannin and LY294002 abrogated the inhibitory effect of DMPP on LPS-induced TNF release by monocytes. Treatment with DMPP for 24 and 48 h provoked a mild PLC phosphorylation, which was blocked by the nAChR antagonist mecamylamine and reversed by PI3K inhibitors. Treatment of monocytes and alveolar macrophages with DMPP reduced the inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium mobilization induced by platelet-activating factor (PAF), an effect that was reversed by mecamylamine in alveolar macrophages. DMPP did not have any effect on PAF receptor expression. DMPP also inhibited the thapsigargin-provoked calcium release, indicating that the endoplasmic reticulum calcium stores might be depleted by treatment with the nAChR agonist. Taken together, these results suggest that PI3K and PLC activation is involved in the anti-inflammatory effect of DMPP. PLC limited, but constant activation could induce, the depletion of intracellular calcium stores, leading to the anti-inflammatory effect of DMPP.

phosphatidylinositol 3-kinase; intracellular calcium; nicotinic acetylcholine receptors; phospholipase C

NICOTINIC RECEPTOR AGONISTS show anti-inflammatory properties in vivo. Studies have demonstrated that administration of nicotine has anti-inflammatory effects in a mouse model of hypersensitivity pneumonitis, reduces the incidence of type 1 (autoimmune) diabetes and ulcerative colitis (1, 21, 24), and improves survival in experimental sepsis (27). Moreover, dimethylphenylpiperazinium (DMPP), a synthetic nicotinic acetylcholine receptor (nAChR) agonist, reduces airway inflammation as well as the increase in airway resistance in a mouse model of asthma (2). Finally, both nAChR agonists nicotine and DMPP have protective effects on early phase hepatic ischemia-reperfusion injury (6, 27).

These effects are further supported by in vitro studies showing that nicotine reduces the production of IL-1 by macrophages (22), TNF expression and IFN-{gamma} and IL-10 mRNA production by alveolar macrophages (AM) (1). Nicotine also has inhibitory effects on the expression of the co-stimulatory molecules CD28 and CTLA-4 on lymphocytes(15) and CD80 on AM (1). DMPP inhibits TNF and IL-6 production by AM (23) and natural killer (NK) cell activity (8). These anti-inflammatory effects are nAChR specific (23).

The intracellular pathways involved in these anti-inflammatory effects remain poorly understood. Previous studies suggested that intracellular calcium mobilization is reduced by nicotine treatment and that nicotine provokes phosphorylation of total protein tyrosine kinases in lymphocytes (14). Others have shown that activation of the {alpha}7 nAChR subunit was required for the abrogation of TNF release by ACh in macrophages (28). There is also proof that the JAK 2-STAT 3 pathway is involved in the anti-inflammatory effect of nAChR activation (7). However, the molecular mechanisms linking nAChR activation and the overall anti-inflammatory effect of nAChR agonists are still misunderstood. Moreover, the exact role of nAChRs on inflammatory cells remains unclear.

Nicotinic receptors normally act as ligand-gated ion channels. Their stimulation on neuronal cells evokes the entry of Ca2+ and Na+ ions (16). However, because inflammatory cells are not excitable cells, their depolarization is unlikely to be the mechanism underlying the immunosuppressive effect of nAChR stimulation. A recent study (17) has reported that stimulation of nAChRs triggers intracellular signalization. In fact, it was shown that the {alpha}7 nAChR subunit is associated with phosphatidylinositol 3-kinase (PI3K) in neurons and that PI3K activation is involved in the neuro-protective effect of nicotine (17).

The purpose of the present study was to assess the signaling pathway involved in the decrease of LPS-induced TNF production by DMPP-treated monocytes/macrophages, to evaluate whether the PI3K association with the {alpha}7 nAChR subunit found in neurons (17) is also present in monocytes and to determine the potential involvement of PI3K in the inhibitory effect of DMPP on the release of TNF by monocytes. The effect of nAChR stimulation on phospholipase C (PLC) activation and the effect of DMPP on the intracellular calcium mobilization in monocytes and AM were also studied.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 
Isolation of whole blood monocytes and AM. This project was approved by the Laval Hospital Research Center ethics committee, and all participants signed a consent form. Studies on PI3K, PLC activation, and intracellular calcium were performed on blood monocytes. AM were also used for intracellular calcium studies.

Blood was obtained from healthy donors, and whole blood mononuclear cells were isolated by Ficoll gradient. Cells were plated for 2 h at 37°C in RPMI medium (GIBCO BRL, Burlington, ON, Canada) completed with 2% of the donor's serum to facilitate monocyte adhesion. Plates were washed four times with HBSS (GIBCO BRL). Purified adherent monocytes were incubated with complete RPMI medium for the rest of the experiments.

AM were isolated from bronchoalveolar lavages performed on mild asthmatic patients who were on inhaled beta-agonists on a prescribe-as-needed (PRN) basis only. Cells were plated and incubated for 2 h at 37°C. Plates were washed four times with HBSS, and purified adherent macrophages were incubated with complete RPMI medium for the subsequent experiments.

Evaluation of TNF production by monocytes. As previously described (23), monocytes (3 x 105) were stimulated with 50 ng of Escherichia coli LPS for 24 h and simultaneously treated, or not, with 10, 20, and 40 µM DMPP. DMPP-treated cells were preincubated (or not) with the PI3K inhibitors wortmannin (100 nM) and LY294002 (10 µM) (both from Sigma, Oakville, ON, Canada) for 3 h. PI3K inhibitor concentrations used in this study were previously described to specifically inhibit PI3K (4, 13, 29). The production of TNF in the supernatant was measured by ELISA (R&D Systems, Minneapolis, MN).

Co-immunoprecipitation of PI3K and nAChR {alpha}3, -4, and -7 subunits. Monocytes (5 x 106) were lysed with lysis buffer [10 mM Tris·HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2% Triton X-100, 1% mixture of protease inhibitors (2 mM EDTA, 2 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin; all from Sigma)] and incubated with Sepharose A gel beads coupled to a rabbit anti-human PI3K P85 (Santa Cruz Biotechnology, Santa Cruz, CA) for 24 h at 4°C. Immunoprecipitates were run in 10% SDS-PAGE, and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were cut and incubated with rabbit anti-human {alpha}3, -4, and -7 nAChR subunits (Santa Cruz Biotechnology) followed by incubation with a polyclonal goat-anti rabbit Ig-coupled to horseradish peroxidase (BD Pharmingen, Missisauga, ON, Canada). Final revelation was accomplished by chemiluminescence with enhanced chemiluminescence (ECL; Amersham Biosciences, Montreal, QC, Canada). Membranes were stripped and reprobed with the rabbit anti-human PI3K to confirm the presence of PI3K in the co-immunoprecipitates. An irrelevant antibody (rabbit Ig) was used as negative control in the immunoprecipitation; membranes were cut and probed with the four previously used antibodies to check for any nonspecific binding.

Immunoprecipitation of PLC and PLC phosphorylation. Monocytes (5 x 106) were treated with 40 µM DMPP for 24 or 48 h, with or without the addition of 10 µM nAChR antagonist mecamylamine (Sigma). Cells were lysed in lysis buffer (described above) complemented with phosphatase inhibitors NaF (0.4 mg/ml) and sodium orthovanadate (0.35 mg/ml). Lysates were incubated with Sepharose A gel beads coupled to a rabbit anti-human PLC for 24 h. Immunoprecipitates were run in 10% SDS-PAGE, and proteins were transferred to a PVDF membrane. Membranes were incubated with a rabbit anti-human phosphotyrosine (Santa Cruz Biotechnology) followed by a goat anti-rabbit Ig-coupled to horseradish peroxidase (BD Pharmingen). Final revelation was achieved by chemiluminescence with ECL (Amersham Biosciences). Membranes were stripped and reprobed with the rabbit anti-human PLC to confirm the initial amount of PLC present in each sample. The intensity of the bands was quantified using Scion-Image software.

Intracellular calcium determination. AM or monocytes (200,000) were plated in a 2.5-cm-diameter petri dish and incubated with 2.5 µM fura-2-AM for 30 min at 37°C. Cells were rinsed to remove extra cellular fura-2. Changes in intracellular calcium concentration ([Ca2+]i) were followed by fura-2 fluorescence, using the Imagemaster system (Photon Technology International, Monmouth Junction, NJ) coupled to a Leica DM IRB fluorescence microscope (x40) (Leica, St-Laurent, QC, Canada). Excitation wavelengths were 353 and 374 nm, and the emission wavelength was 510 nm. The ratio between the fluorescence provoked by the 353- and the 374-nm excitation wavelengths was calculated and used as [Ca2+]i indicator.

Cells were pretreated, or not, for 24 h with 160 µM DMPP and stimulated with either 10 µM platelet-activating factor (PAF) or 1 mM thapsigargin (both from Sigma) in calcium buffer. Fluorescence at 510 nm was recorded every 20 s for 20 min. To verify the specificity of the response to nAChRs, the experiments on the effect of DMPP on PAF-activated AM were repeated in the presence of 10 µM mecamylamine, a nicotinic receptor antagonist.

Cell ELISA for PAF receptor expression. The expression of PAF receptor (PAFr) was assessed by cell ELISA. Monocytes (1 x 105 per well) were plated onto 96-well plates and pretreated, or not, with 160 µM DMPP. Twenty-four hours later, cells were washed with PBS and adsorbed onto 96-well plates by evaporation overnight at room temperature. The cells were then fixed with methanol at –20°C for 20 min. Endogenous peroxidase and pseudoperoxidase activities were blocked with DAKO peroxidase-blocking reagent. Cells were blocked with 1% BSA before incubation with 5 µg/ml rabbit anti-PAFr (Cayman Chemical, Hornby, ON, Canada) or rabbit IgG (Cayman Chemical) as isotype control. Binding was revealed with peroxidase-conjugated goat anti-rabbit IgG (H+L) (Cedarlane, Hornby, ON, Canada) followed by the addition of a substrate solution (R&D Systems, Hornby, ON, Canada). The reaction was stopped after 3 min with H2SO4 (2 N), and optical density (OD) was read at 450 nm with correction at 550 nm (Molecular Devices, microplate reader). OD from isotype control was subtracted from that of PAFr-tested wells.

Statistical analysis. Statistical analysis for the TNF production was made with the use of an ANOVA table followed by a Fishers protected least significant differences post hoc test. Differences in PLC phosphorylation were analyzed by a paired t-test, and analyses for the calcium studies and cell ELISA were made using an unpaired t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 
PI3K activity is involved in the anti-inflammatory effect of DMPP. The involvement of PI3K on the anti-inflammatory effect of DMPP in monocytes was tested by use of the PI3K inhibitors wortmannin and LY294002. Cell viability was not affected by DMPP (data not shown). A previous dose response with DMPP (range, 0.1 to 320 µM) had shown that DMPP significantly inhibits TNF production by monocytes, starting at 10 µM, and that doses >40 µM provoke no further decreasing effect (data not shown). Therefore, the 10, 20, and 40 µM doses were used here to test the involvement of PI3K in this anti-inflammatory effect. Again, in these experiments, treatment of monocytes for 24 h with 10, 20, and 40 µM DMPP dose dependently inhibited TNF release (Fig. 1) (23). Pretreatment of monocytes with the PI3K inhibitors wortmannin and LY294002 partially reversed the inhibitory effect of DMPP on TNF release. The association of nAChR with PI3K was tested by co-immunoprecipitation. Figure 2 shows that PI3K is associated with {alpha}3, {alpha}4, and {alpha}7 nAChR subunits.


Figure 1
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 1. Effect of phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 on TNF production by monocytes. Experiment was repeated with monocytes from 7 different donors. LPS-induced TNF release in monocytes was significantly blocked by dimethylphenylpiperazinium (DMPP) (P < 0.05 for all DMPP doses). Pretreatment of monocytes with the PI3K inhibitor wortmannin significantly reversed the 40 µM DMPP dose (P = 0.04), and the PI3K inhibitor LY294002 significantly reversed the inhibitory effect at all DMPP doses (P ≤ 0.008). *Significant result.

 

Figure 2
View larger version (57K):
[in this window]
[in a new window]
  		Fig. 2. Association of PI3K with nicotinic acetylcholine receptors (nAChRs). Co-immunoprecipitation of {alpha}3, {alpha}4, and {alpha}7 subunits and PI3K (P85) shows that all 3 subunits were physically associated with PI3K in monocytes. P85 band was revealed after stripping of the membranes as proof that PI3K was indeed present in the co-immunoprecipitates. Bottom blot shows the negative control in which an irrelevant antibody (rabbit Ig) was used for the immunoprecipitation. Membranes were cut, and the 4 antibodies used in the co-immunoprecipitation (anti-nAChR {alpha}3, -4, and -7 subunits and anti-P85) showed no nonspecific reactivity. These are representative results of 3 different experiments.

 
PLC is phosphorylated by DMPP treatment. Treatment of monocytes with 40 µM DMPP for 24 and 48 h provoked the phosphorylation of PLC as demonstrated by immunoprecipitation of PLC and revelation of phospho-PLC. This effect was blocked by adding mecamylamine 5 min before the start of the DMPP treatment (Fig. 3A). This effect was also partially reversed by addition of the PI3K inhibitors wortmannin and LY294002 (Fig. 3B). Analysis of the intensity of the bands confirmed that both wortmannin and LY294002 significantly reversed the level of phospho-PLC compared with the DMPP-treated cells (Fig. 3C).


Figure 3
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 3. Effect of DMPP treatment on phospholipase C (PLC) phosphorylation. These are representative results of 3 different experiments. Revelation of PLC after membrane stripping shows that the initial amount of PLC was constant in each condition. A: immunoprecipitation of PLC and revelation of phospho-PLC showed that stimulation of monocytes with 40 µM DMPP for 24 and 48 h provoked a mild PLC phosphorylation, which was inhibited by the nAChR antagonist mecamylamine. B and C: PLC phosphorylation provoked by 24-h DMPP treatment (160 µM) was also significantly reversed by the addition of the 2 PI3K inhibitors wortmannin and LY294002 (n = 3).

 
DMPP blocks intracellular calcium increase provoked by PAF. All intracellular calcium-related results are presented as a representative result of three to five different experiments, with each experiment consisting of a simultaneous recording of fluorescence ratios from three to five cells.

Intracellular calcium rise in monocytes (Fig. 4A) or human AM (Fig. 4B) was induced by PAF. This molecule is known to stimulate intracellular calcium mobilization in those cells (20). Cell stimulation was achieved after an 8- to 10-min resting period. Recording of fura-2 fluorescence was stopped after 10 min of cell stimulation, when the reaction had reached a plateau.


Figure 4
View larger version (7K):
[in this window]
[in a new window]
  		Fig. 4. Effect of DMPP on the intracellular calcium rise provoked by the addition of platelet-activating factor (PAF) in monocytes (A) and alveolar macrophages (AM) (B). Stimulation of the G protein-coupled PAF receptor provoked a quick initial peak [inositol 1,4,5-trisphosphate (IP3)-dependent calcium increase] followed by a more sustained response (store-operated calcium entry). DMPP treatment in monocytes reduced the initial peak by ~30% and significantly delayed the response to PAF. In AM, the intensity of the initial peak was reduced by 50% in DMPP-treated cells. C: expression of PAF receptor (PAFr) was not affected by the treatment with 160 µM DMPP. Each column represents the mean ± SE for purified blood monocytes from 4 subjects (3 repeats by subject). OD, optical density.

 
In DMPP-treated, PAF-stimulated monocytes (Fig. 4A), the intensity of the initial peak of internal calcium concentration [corresponding to the inositol 1,4,5-trisphosphate (IP3)-dependant calcium rise provoked by the stimulation of the G protein-coupled PAFr] (9) was decreased by 33.4 ± 2.6% (n = 14, P = 0.03) and delayed by 235.8 ± 41.0 s (n = 14, P < 0.0001) compared with nontreated cells. In AM, DMPP treatment reduced the initial peak by 51.7 ± 7.7% (n = 18, P < 0.0001). Different timings of incubation (10 min and 1, 2, 24, and 48 h; data not shown) with DMPP were tested. Both 24- and 48-h DMPP treatments were the most effective in blocking the intracellular calcium rise provoked by PAF; 24-h incubation was therefore used for all calcium studies. The cell ELISA method was used to verify whether the downregulating effect on calcium mobilization could be due to reduced PAFr expression in monocytes. As shown in Fig. 4C, cells incubated with or without DMPP had similar PAFr expression. Mecamylamine, an nAChR antagonist, blunted the inhibitory effect of DMPP on the PAF-induced intracellular calcium rise in AM (Fig. 5).


Figure 5
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 5. Effect of nAChR antagonist mecamylamine on the inhibition of calcium increase by DMPP. The nAChR antagonist mecamylamine (10 µM) was added right before (5 min) DMPP and blunted the blocking effect of DMPP on the increase in intracellular calcium provoked by PAF.

 
DMPP reduces the IP3-dependent increase in intracellular calcium. In experiments performed in the absence of extracellular calcium in AM, PAF provoked a quick intracellular calcium increase (IP3-dependant calcium increase), and the sustained response provoked by PAF in the presence of extracellular calcium was not observed. Even in the absence of extracellular calcium, DMPP blocked the response of AM to PAF (Fig. 6), indicating that DMPP has a direct effect on this phase of calcium rise in the cells. DMPP reduced the intensity of the peaks by 56.7 ± 11.5% (n = 9, P = 0.0017).


Figure 6
View larger version (8K):
[in this window]
[in a new window]
  		Fig. 6. Effect of DMPP on calcium mobilization in calcium-free medium. Stimulation of AM with PAF in calcium-free medium provoked a quick calcium increase (IP3 dependent), which was blocked by 56.7 ± 11.5% in DMPP-treated cells.

 
DMPP depletes IP3-dependent intracellular calcium stores in AM. Addition of thapsigargin in AM, which inactivates the ATPase calcium pump, provoked a cytoplasmic calcium increase (Fig. 7) corresponding to the depletion of endoplasmic reticulum calcium stores. Pretreatment for 24 h with 160 µM DMPP reduced the response provoked by thapsigargin, indicating that the pretreatment of cells with DMPP had depleted IP3-dependent intracellular calcium stores.


Figure 7
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 7. Effect of DMPP on thapsigargin-induced depletion of the endoplasmic reticulum calcium content in AM. The increase in intracellular calcium induced by the addition of thapsigargin had a smaller amplitude in DMPP-treated cells than in nontreated cells, indicating that DMPP depleted intracellular calcium stores.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 GRANTS
 REFERENCES
 
The link between direct activation of nAChRs and the inhibitory effect of nicotine on intracellular calcium dynamics in general immune cell function is still being elucidated. Because the association studies were conducted by co-immunoprecipitation, we cannot rule out the possibility that the interaction between PI3K P85 and {alpha}3, -4, and -7 nAChR subunits could involve another protein. These results suggested that PI3K could be activated by nAChR activation by DMPP. This was confirmed by the reversal of the downregulating effect of DMPP on monocyte TNF production by the PI3K inhibitors wortmannin and LY294002. The fact that these inhibitors did not completely reverse the anti-inflammatory effect of DMPP suggests that other signaling pathways may be involved. The difference in the response of DMPP-treated monocytes to the two P13K inhibitor is surprising. However, it has been reported that LY294002 and wortmannin inhibit PI3K by different mechanisms; LY294002 acts as a pure competitive inhibitor of ATP, whereas wortmannin alkalizes a lysine residue at the ATP-binding site (26). There is also evidence that LY294002 could inhibit other kinases such as caseine kinase-2, whereas wortmannin is more PI3K specific (26). The different mechanisms of PI3K inhibition could explain the differences in the response of DMPP-treated monocytes to these two molecules. Nevertheless, these two inhibitors are often used in parallel to verify the implication of PI3K activation in a variety of phenomena (4, 10, 13, 29). We therefore believe that the effect reported here is PI3K specific.

PI3K partially activates PLC in monocytes/macrophages (11). We believe that PLC phosphorylation induced by nAChR activation could be due to PI3K activation via nAChR stimulation. Blockade of PLC phosphorylation by the nAChR general antagonist mecamylamine indicates that this phenomenon is specific to nAChR activation. Partial reversal of the DMPP-induced PLC phosphorylation by PI3K inhibitors also suggests that this is mediated, at least in part, by PI3K activation. Because PLC is a major player in intracellular calcium mobilization from the endoplasmic reticulum, a constant and mild PLC phosphorylation by nAChR agonists, such as that provoked by DMPP, could influence the intracellular calcium mobilization in monocytes/macrophages. We are aware that PLC phosphorylation is not a direct proof of protein activation. However, because it is widely established that PLC phosphorylation correlates its activity (18), and that the antibody used in our study was previously used to describe PLC activity (30, 31), we believe that, because DMPP provokes PLC phosphorylation, it should promote its activation.

The blockade by DMPP of the PAF-stimulated quick rise in intracellular calcium in the absence of extracellular calcium further confirms that DMPP affects the PLC/IP3-dependant intracellular calcium rise. This hypothesis is also supported by the reduced effect of DMPP on thapsigargin-induced depletion of endoplasmic reticulum calcium stores. Taken together, these results suggest that the overall reducing effect of DMPP on the intracellular calcium increase provoked by PAF in monocytes and AM could come from a depletion of these stores. Depletion of calcium stores by nicotine, another nicotinic agonist, was previously reported in lymphocytes (25). However, it is well known that depletion of intracellular calcium stores normally provokes opening of store-operated channels (3). DMPP could also affect the opening of these channels, since it appears that the content in intracellular calcium could not be renewed after intracellular store depletion.

Although it is known that PLC phosphorylation leads to calcium efflux from the endoplasmic reticulum, and that DMPP provoked a mild PLC phosphorylation, addition of DMPP by itself did not create any detectable calcium mobilization. Moreover, in preliminary studies using human AM from hypersensitivity pneumonitis patients, the longer cells were treated with DMPP, the more the intracellular calcium mobilization was reduced (data not shown). We believe that this further supports the hypothesis that the DMPP-provoked PLC phosphorylation could, in chronically treated cells, lead to an intracellular calcium efflux from the endoplasmic reticulum without reaching the intracellular calcium level needed to provoke calcium-induced intracellular signaling or renewal of calcium store content, leading to the depletion of the endoplasmic reticulum calcium stores.

Experiments showing that the nicotinic antagonist mecamylamine abrogated the inhibitory effect of DMPP on the intracellular calcium rise in PAF-stimulated AM confirm that the observed phenomenon is specific to nAChR activation. We verified whether the downregulating effect of DMPP on intracellular calcium mobilization could be due to a reduced PAF receptor expression. Results suggest that PAF receptor expression was not modulated by DMPP, which further confirms that the inhibitory effect of this nAChR agonist on the intracellular calcium mobilization is nAChR specific. We do not know which specific nAChR subunit is implicated in these effects. The determination of which specific subunit is involved in the general anti-inflammatory effect of DMPP could be of interest to develop a potential treatment of inflammatory diseases by activators of specific nicotinic receptor subunits.

The link between the PI3K and PLC results and the intracellular calcium observation is not obvious because of the use of two different cell types (monocytes and alveolar macrophages) as well as two different stimulants (LPS and PAF). LPS is known to induce TNF release (12, 19, 32). We failed to induce intracellular calcium mobilization in AM by LPS (data not shown). Because PAF is known to induce intracellular calcium mobilization in macrophages (20) and is involved in the overall stimulatory effect of LPS on macrophages (5), this molecule seemed like the obvious stimulant to use in the intracellular calcium studies. The fact that the overall inhibitory effect of DMPP on the intracellular calcium mobilization was observed in both monocytes and AM strengthens the link between the two different cell types used in this study and strongly suggests that the intracellular calcium store depletion observed in DMPP-treated AM should occur in monocytes as well.

Our general hypothesis explaining the link between nAChR activation and the anti-inflammatory effect of DMPP is that chronic nicotinic receptor activation by DMPP activates PI3K, which in turn partially activates PLC. PLC is responsible for IP3 production, whose limited but constant presence in the cytoplasm could deplete intracellular calcium stores. Intracellular calcium store depletion could in term lead to impaired monocyte/macrophage activation.

In conclusion, the anti-inflammatory effect of the nicotinic agonist DMPP is mediated by PI3K activation. Activation of nAChRs induces PLC phosphorylation, which could lead to intracellular calcium store depletion. A better understanding of the intracellular pathways underlying the anti-inflammatory effect of nicotinic agonists is another step toward treating inflammatory diseases with this family of molecules.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
GRANTS
 REFERENCES
 
M.-R. Blanchet and E. Israël-Assayag are co-authors of the patent no. PCT/CA02/00412 (March 2002), which is owned by Laval University. Y. Cormier is the principal author of the patent no. PCT/CA02/00412.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
REFERENCES
 
This work was funded by the Institut de Recherche Robert-Sauvé en Santé et Sécurité au Travail and by the Canadian Institute for Health Research.


   ACKNOWLEDGMENTS
 
We thank Dominique Fournier for help with the calcium studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Cormier, Centre de Pneumologie, Hôpital Laval, 2725 Chemin Ste-Foy, Ste-Foy, Québec, Canada G1V 4G5 (e-mail: yvon.cormier{at}med.ulaval.ca)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 GRANTS
 REFERENCES
 

  1. Blanchet MR, Israel-Assayag E, and Cormier Y. Inhibitory effect of nicotine on experimental hypersensitivity pneumonitis in vivo and in vitro. Am J Respir Crit Care Med 169: 903–909, 2004.[Abstract/Free Full Text]
  2. Blanchet MR, Israel-Assayag E, and Cormier Y. Modulation of airway inflammation and resistance in mice by a nicotinic receptor agonist. Eur Respir J 26: 21–27, 2005.[Abstract/Free Full Text]
  3. Bolotina VM. Store-operated channels: diversity and activation mechanisms. Sci STKE 243: e34, 2004.
  4. Boudot C, Kadri Z, Petitfrere E, Lambert E, Chretien S, Mayeux P, Haye B, and Billat C. Phosphatidylinositol 3-kinase regulates glycosylphosphatidylinositol hydrolysis through PLC-gamma(2) activation in erythropoietin-stimulated cells. Cell Signal 14: 869–878, 2002.[CrossRef][Web of Science][Medline]
  5. Bulger EM, Arbabi S, Garcia I, and Maier RV. The macrophage response to endotoxin requires platelet activating factor. Shock 17: 173–179, 2002.[CrossRef][Web of Science][Medline]
  6. Crockett ET, Galligan JJ, Uhal BD, Harkema J, Roth R, and Pandya K. Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists. BMC Clin Pathol 6: 3, 2006.[CrossRef][Medline]
  7. De Jonge WJ, van der Zanden EP, The FO, Bijlsma MF, van Westerloo DJ, Bennink RJ, Berthoud HR, Uematsu S, Akira S, van den Wijngaard RM, and Boeckxstaens GE. Stimulation of the vagus nerve attenuates macrophage activation by activating the Jak2-STAT3 signaling pathway. Nat Immun 6: 844–851, 2005.[Medline]
  8. Fecho K, Maslonek KA, Dykstra LA, and Lysle DT. Alterations of immune status induced by the sympathetic nervous system: immunomodulatory effects of DMPP alone and in combination with morphine. Brain Behav Immun 7: 253–270, 1993.[CrossRef][Web of Science][Medline]
  9. Galione A and Churchill GC. Interactions between calcium release pathways: multiple messengers and multiple stores. Cell Calcium 32: 343–354, 2002.[CrossRef][Web of Science][Medline]
  10. Herrera-Velit P, Knutson KL, and Reiner NE. Phosphatidylinositol 3-kinase-dependent activation of protein kinase C-zeta in bacterial lipopolysaccharide-treated human monocytes. J Biol Chem 272: 16445–16452, 1997.[Abstract/Free Full Text]
  11. Hiller G and Sundler R. Regulation of phospholipase C-gamma 2 via phosphatidylinositol 3-kinase in macrophages. Cell Signal 14: 169–173, 2002.[CrossRef][Web of Science][Medline]
  12. Honma K, Udono H, Kohno T, Yamamoto K, Ogawa A, Takemori T, Kumatori A, Suzuki S, Matsuyama T, and Yui K. Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS. Proc Natl Acad Sci USA 102: 16001–16006, 2005.[Abstract/Free Full Text]
  13. Jinnin M, Ihn H, Asano Y, Yamane K, Trojanowska M, and Tamaki K. Platelet derived growth factor induced tenascin-C transcription is phosphoinositide 3-kinase/Akt-dependent and mediated by Ets family transcription factors. J Cell Physiol 206: 718–727, 2006.[CrossRef][Web of Science][Medline]
  14. Kalra R, Singh SP, Kracko D, Matta SG, Sharp BM, and Sopori ML. Chronic self-administration of nicotine in rats impairs T cell responsiveness. J Pharmacol Exp Ther 302: 935–939, 2002.[Abstract/Free Full Text]
  15. Kalra R, Singh SP, Savage SM, Finch GL, and Sopori ML. Effects of cigarette smoke on immune response: chronic exposure to cigarette smoke impairs antigen-mediated signaling in T cells and depletes IP3-sensitive Ca(2+) stores. J Pharmacol Exp Ther 293: 166–171, 2000.[Abstract/Free Full Text]
  16. Katzung BG. Basic and clinical pharmacology. New York: Mc Graw-Hill/Appleton and Lange, 1998.
  17. Kihara T, Shimohama S, Sawada H, Honda K, Nakamizo T, Shibasaki H, Kume T, and Akaike A. alpha 7 nicotinic receptor transduces signals to phosphatidylinositol 3-kinase to block A beta-amyloid-induced neurotoxicity. J Biol Chem 276: 13541–13546, 2001.[Abstract/Free Full Text]
  18. Kim HK, Kim JW, Zilberstein A, Margolis B, Kim JG, Schlessinger J, and Rhee SG. PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. Cell 65: 435–441, 1991.[CrossRef][Web of Science][Medline]
  19. Laan TT, Bull S, Pirie RS, and Fink-Gremmels J. Evaluation of cytokine production by equine alveolar macrophages exposed to lipopolysaccharide, Aspergillus fumigatus, and a suspension of hay dust. Am J Vet Res 66: 1584–1589, 2005.[CrossRef][Web of Science][Medline]
  20. Lendvai B, Santha E, Szelenyi J, and Hasko G. Platelet-activating factor evokes Ca2+ transients after the blockade of ryanodine receptor by dantrolene in RAW 264.7 macrophages. Neurochem Res 26: 1007–1013, 2001.[CrossRef][Web of Science][Medline]
  21. Mabley JG, Pacher P, Southan GJ, Salzman AL, and Szabo C. Nicotine reduces the incidence of type I diabetes in mice. J Pharmacol Exp Ther 300: 876–881, 2002.[Abstract/Free Full Text]
  22. Madretsma S, Wolters LM, van Dijk JP, Tak CJ, Feyerabend C, Wilson JH, and Zijlstra FJ. In-vivo effect of nicotine on cytokine production by human non-adherent mononuclear cells. Eur J Gastroenterol Hepatol 8: 1017–1020, 1996.[Web of Science][Medline]
  23. Matsunaga K, Klein TW, Friedman H, and Yamamoto Y. Involvement of nicotinic acetylcholine receptors in suppression of antimicrobial activity and cytokine responses of alveolar macrophages to Legionella pneumophila infection by nicotine. J Immunol 167: 6518–6524, 2001.[Abstract/Free Full Text]
  24. Sandborn WJ. Nicotine therapy for ulcerative colitis: a review of rationale, mechanisms, pharmacology, and clinical results. Am J Gastroenterol 94: 1161–1171, 1999.[CrossRef][Web of Science][Medline]
  25. Sopori ML, Kozak W, Savage SM, Geng Y, Soszynski D, Kluger MJ, Perryman EK, and Snow GE. Effect of nicotine on the immune system: possible regulation of immune responses by central and peripheral mechanisms. Psychoneuroendocrinology 23: 189–204, 1998.[CrossRef][Web of Science][Medline]
  26. Stein RC. Prospects for phosphoinositide 3-kinase inhibition as a cancer treatment. Endocr Relat Cancer 8: 237–248, 2001.[Abstract]
  27. Wang H, Liao H, Ochani M, Justiniani M, Lin X, Yang L, Al Abed Y, Wang H, Metz C, Miller EJ, Tracey KJ, and Ulloa L. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med 10: 1216–1221, 2004.[CrossRef][Web of Science][Medline]
  28. Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Wang H, Yang H, Ulloa L, Al Abed Y, Czura CJ, and Tracey KJ. Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature 421: 384–388, 2003.[CrossRef][Medline]
  29. Wynne AM, Mocanu MM, and Yellon DM. Pioglitazone mimics preconditioning in the isolated perfused rat heart: a role for the prosurvival kinases PI3K and P42/44MAPK. J Cardiovasc Pharmacol 46: 817–822, 2005.[CrossRef][Web of Science][Medline]
  30. Zapf-Colby A, Eichhorn J, Webster NJ, and Olefsky JM. Inhibition of PLC-gamma1 activity converts nerve growth factor from an anti-mitogenic to a mitogenic signal in CHO cells. Oncogene 18: 4908–4919, 1999.[CrossRef][Web of Science][Medline]
  31. Zhang X, Chattopadhyay A, Ji QS, Owen JD, Ruest PJ, Carpenter G, and Hanks SK. Focal adhesion kinase promotes phospholipase C-gamma1 activity. Proc Natl Acad Sci USA 96: 9021–9026, 1999.[Abstract/Free Full Text]
  32. Zhou H, Ding GF, Liu W, Wang LX, Lu YL, Cao HW, and Zheng J. Lipopolysaccharide could be internalized into human peripheral blood mononuclear cells and elicit TNF-{alpha} release, but not via the pathway of toll-like receptor 4 on the cell surface. Cell Mol Immunol 1: 373–377, 2004.[Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
M.-R. Blanchet, A. Langlois, E. Israel-Assayag, M.-J. Beaulieu, C. Ferland, M. Laviolette, and Y. Cormier
Modulation of eosinophil activation in vitro by a nicotinic receptor agonist
J. Leukoc. Biol., May 1, 2007; 81(5): 1245 - 1251.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/4/L757    most recent
00409.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (5)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Blanchet, M.-R.
Right arrow Articles by Cormier, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Blanchet, M.-R.
Right arrow Articles by Cormier, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.