Cytokine production by alveolar macrophages is down regulated by the {alpha}-methylhydroxylation pathway of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Léa-Isabelle Proulx1, André Castonguay2 and Elyse Y. Bissonnette1,3

1 Centre de Recherche, Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie de l'Université Laval, Canada and 2 Faculté de Pharmacie, Université Laval, Hôpital Laval, 2725, Chemin Ste-Foy, Ste-Foy, QC, G1V 4G5, Canada

3 To whom correspondence should be addressed Email: elyse.bissonnette{at}med.ulaval.ca


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
NNK, a nicotine-derived nitrosamine, is a potent lung carcinogen that generates electrophilic intermediates capable of damaging DNA. The effects of NNK on the immune response, which may facilitate lung carcinogenesis, are poorly understood. Alveolar macrophages (AM), a key cell in the maintenance of lung homeostasis, metabolize NNK via two major metabolic activation pathways: {alpha}-methylhydroxylation and {alpha}-methylenehydroxylation. We have shown previously that NNK inhibits the production of interleukin-12 (IL-12) and tumor necrosis factor (TNF), but stimulates the production of IL-10 and prostaglandin E2 (PGE2) by AM. In the present study, we investigated the contribution of each activation pathway in the modulation of AM function. We used two precursors, 4-[(acetoxymethyl)-nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) and N-nitro(acetoxymethyl)methylamine (NDMAOAc), which generate the reactive electrophilic intermediates [4-(3-pyridyl)-4-oxo-butanediazohydroxide and methanediazohydroxide, respectively] in high yield and exclusively. Rat AM cell line, NR8383, was stimulated and treated with different concentrations of NNKOAc or NDMAOAc (12, 25 and 50 µM). Mediator release was measured in cell-free supernatants. NNKOAc significantly inhibited the production of IL-10, IL-12, TNF and nitric oxide but increased the release of PGE2 and cyclooxygenase-2 expression suggesting that the {alpha}-methylhydroxylation pathway might be responsible for NNK modulation of AM cytokine release. In contrast, NDMAOAc did not modulate AM mediator production. However, none of these precursors, alone or in combination, could explain the stimulation of AM IL-10 production by NNK. Our results suggest that the {alpha}-methylhydroxylation of NNK leading to DNA pyridyloxobutylation also modulates cytokine production in NNK-treated AM.

Abbreviations: AM, alveolar macrophages; BCG, Bacille Calmette-Guerin; COX, cyclooxygenase; IL, Interleukin; KA, keto acid; KAL, keto alcohol; LPS, lipopolysaccharide; NDMAOAc, nitroso(acetoxymethyl)methylamine; NO, nitric oxide; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNKOAc, 4-(acetoxy-methylnitrosamino)-1-(3-pyridyl)-1-butanone; PGE2, prostaglandin-E2


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Lung cancer is one of the leading causes of cancer death in Canada and the US (1,2). Cigarette smoking is responsible for 90 and 80% lung cancer in men and women, respectively (35). Cigarette smoke contains more than 4000 components with 55 known as being carcinogenic. Among those, the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) derived from nicotine is one of the most important considering the amount present in cigarette smoke (1). Furthermore, previous studies have shown that NNK has a remarkable specificity for pulmonary tissues suggesting a significant role of NNK in the high incidence of lung cancer in cigarette smokers (69).

NNK needs to be enzyme-activated to exert its carcinogenic effect (3,7,8). Three metabolic activation pathways of NNK have been identified (8,10,11). Two metabolic activation pathways involving hydroxylation of the carbons adjacent to the N-nitroso group have been identified. The {alpha}-methylhydroxylation and the {alpha}-methylenehydroxylation, catalyzed by cytochrome P450, lipoxygenases and cyclooxygenases (COX) (10,12), lead to formation of the two reactive intermediates, 4-(3-pyridyl)-4-oxo-butanediazohydroxide and methanediazohydroxide, respectively (8,10,11). These intermediates can pyridyloxobutylate and methylate DNA (Figure 1) (13). Moreover, reduction of the carbonyl group of NNK rapidly yields 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a circulating metabolite in smokers formed in part in human lung (1315). The carcinogenic activities of NNAL and NNK are similar (10). Although the carcinogenesis mechanisms of NNK are well documented (10), few studies have investigated its modulation of the immune response.



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Fig. 1. {alpha}-Hydroxylation activation pathway of NNK and precursor activation pathways.

 
Alveolar macrophages (AM) are located at the interface air tissues in alveoli and in the conducting airways. They are the first cells to come into contact with foreign particles and are responsible for maintaining lung homeostasis by stimulating or inhibiting inflammatory responses (16). AM produce several mediators important for host defence such as interleukin-12 (IL-12), tumor necrosis factor (TNF) and nitric oxide (NO). IL-12 is an inflammatory cytokine known to activate the cytotoxicity of AM, lymphocytes and natural killer cells (1719), whereas TNF and NO are two mediators implicated in inflammation and cytotoxicity against tumor cells (2025). AM also produce anti-inflammatory mediators such as IL-10 and prostaglandin-E2 (PGE2) that are potent inhibitors of AM functions and inflammatory cytokine production (26,27). Thus, alterations of AM functions might contribute to an increase in the incidence of lung cancer and lung infection observed in smokers.

Given the importance of AM in host defence and their role in lung homeostasis, we investigated the effect of NNK on AM-mediator release. We have demonstrated that NNK inhibits in a concentration- and time-dependent manner the production of IL-12, TNF and NO but stimulates IL-10 and PGE2 release (28). These results may explain the contribution of this cigarette smoke component in the modulation of the immune response observed in smokers and in people exposed to second-hand smoke. However, it was not clear which one of the two types of the {alpha}-hydroxylation pathways was responsible for these effects. Thus, we used two precursors of a reactive intermediate to discriminate the involvement of each pathway in the immunomodulation of AM functions (Figure 1). These precursors, 4-(acetoxy-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) and nitroso(acetoxymethyl)methylamine (NDMAOAc), are not analogous structures of NNK, but they generate the metabolite responsible for NNK DNA pyridyloxobutylation and methylation, respectively. Furthermore, the immunomodulatory effect of the end products of these pathways, keto acid and keto alcohol, were investigated. These products have no carcinogenic activity.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
NNK (99% pure) was purchased from Chemsyn Science Laboratories (Lenexa, KS). NNK metabolites (keto acid and keto alcohol) and NNKOAc were synthesized as described previously (29,30). NDMAOAc and NNAL were purchased from LKT Laboratories (St Paul, MN) and Toronto Research Chemicals (Toronto, ON, Canada), respectively.

Cell culture
NR8383, is an AM cell line, isolated from Sprague–Dawley rats used previously in vitro to study macrophage-related activities (31) and we have shown that functions of NR8383 and human AM are similarly modulated (32). NR8383 were maintained in culture as described previously (31). All experiments were done in RPMI-1640 medium with 5% fetal bovine serum, 1% HEPES buffer, 1% penicillin-streptomycin and 0.2% gentamicin (Invitrogen, Burlington, ON, Canada). After 2 h adherence in 96-well plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ) at 37°C, AM were treated for 20 or 48 h with NNK (500 µM), keto acid (15 µM), keto alcohol (15 µM), increasing concentrations of NNKOAc (12, 25 and 50 µM), NDMAOAc (12, 25 and 50 µM) or NNAL (500 µM) in the presence of lipopolysaccharide (LPS) (10 ng/ml) (Salmonella enteriditis; Sigma Chemical, St Louis, MO) or Bacille Calmette-Guerin (BCG) (5x106 CFU/ml, Calbiochem, San Diego, CA). Concentrations of keto acid and keto alcohol (end products) were based on NNK metabolism by AM (28). After treatment, cell-free supernatants were recovered and stored at –80°C for subsequent analysis.

HPLC analysis
HPLC was performed with a complete Shimadzu system consisting of an automatic sample injector (model SIL-9A), a programmable pump (model LC-1600), a variable UV detector (SPD-6A), and an integrator Chromatopac C-R5A. Analysis was performed on a 5 µ Beckman® ultrasphere ODS column (4.6 x 250 mm) using UV detection (254 nm). The mobile phase was ammonium acetate 10 mM pH 5.5 with 10% methanol (solvent A) or 40% methanol (solvent B). The solvent program was a 50-min linear gradient 0–100% solvent B and then held at 100% solvent B for 7 min with a flow rate of 1 ml/min. At the end of each run, initial conditions were re-established and the column was equilibrated for 10 min in 100% solvent A with a flow rate of 1.2 ml/min.

Mediator production
Levels of TNF, IL-12 and IL-10 were measured in cell-free supernatants using ELISA (Pharmingen, San Diego, CA) and PGE2 using EIA (Cayman Chemical, Ann Harbor, MI) having a sensitivity of 4, 5, 5 and 7 pg/ml, respectively. As reported in a previous study (28), AM need to be stimulated with LPS or BCG to produce a detectable amount of mediators and the effects of NNK were maximal after 20 h treatment (28). Thus, AM were treated for 20 h in the present study.

Measurement of NO production
AM were treated as mentioned above in the presence of LPS (10 ng/ml) for 48 h. Cell-free supernatants were assayed for NO2 using Griess reaction as described previously (31). NO2 concentration, proportional to optical density at 540 nm, was determined using a Vmax kinetic microplate reader (Thermo Max; Molecular Devices, Menlo Park, CA) with reference to a standard curve (NaNO2).

COX-2 flow cytometry analysis
AM were treated as mentioned previously with NNK and NNKOAc for 1 h, blocked with bovin serum albumin 1% (Sigma) for 30 min and fixed with cytofix/cytoperm Plus Kit (Pharmingen) for 20 min. AM were then incubated with mouse anti-COX-2 polyclonal antibody (Cayman Chemical) for 1 h followed by 1 h incubation with Alexa-conjugated goat anti-mouse IgG antibody (Molecular Probe, Eugene, OR). AM were analyzed by flow cytometry using an EPICS® XL-MCL cytometer (Beckman-coulter, Miami, FL). Acquisition of fluorescence data was gated by forward and side light scatter. Isotypic control, consisting of rat IgG, was run in parallel and subtracted from the results obtained with anti-COX-2.

Statistical analysis
Analysis of variance (ANOVA) combined with Student's t test for paired data was used to compare various treatments. Differences were considered significant when P < 0.05.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Activation pathways implicated in the modulation of inflammatory mediators
To first identify the hydrolysis of the NNKOAc by AM, cells were treated with NNKOAc (50 µM) for 20 h with LPS and supernatants were analyzed by HPLC to determine the presence of keto alcohol, an end product of the {alpha}-hydroxymethyl pathway. Keto alcohol was found in the supernatant (data not shown), indicating that AM can metabolize NNKOAc.

To investigate the activation pathways implicated with NNK, AM were treated with LPS for 20 h in the presence of increasing concentrations of NNKOAc (12, 25 and 50 µM) or NDMAOAc (12, 25 and 50 µM), which are two metabolic precursors inducing the production of metabolites responsible for pyridyloxobutylation and methylation intermediates, respectively. The concentrations of NNKOAc and NDMAOAc selected did not affect cell viability as assessed by trypan blue dye exclusion (>80%, data not shown). To demonstrate further the role of reactive intermediates, AM were treated with the end products of activated pathways, keto acid (15 µM) and keto alcohol (15 µM). These concentrations were determined from HPLC analysis of NNK metabolism by AM as determined previously (28). NNKOAc significantly (P < 0.001) inhibited AM TNF release in a concentration-dependent manner, whereas NDMAOAc and keto acid did not modulate TNF production (Figure 2). Interestingly, keto alcohol significantly (P < 0.005) inhibited AM TNF release (27%). These results suggest that the pathway leading to pyridyloxobutylation as well as its end product, keto alcohol, may be responsible for TNF inhibition caused by NNK activation. In contrast, the pathway implicated in DNA methylation and leading to the end product keto acid has no effect on the modulation of TNF production.



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Fig. 2. Modulation of AM TNF production. AM were treated for 20 h with NNK and different concentrations of NNKOAc and NDMAOAc in the presence of LPS (10 ng/ml). NNK and NNKOAc significantly ({dagger}P < 0.001, {ddagger}P < 0.01) inhibited TNF production whereas NDMDOAc, keto alcohol (KAL), and keto acid (KA) had no effect. Means ± SEM of seven experiments.

 
NNK activation pathways implicated in the modulation of AM IL-12 production were also investigated. AM were treated as described above and stimulated with BCG (5 x 106 CFU/ml) to stimulate IL-12 release in supernatants. NNKOAc significantly (P < 0.05) inhibited AM IL-12 release in a concentration-dependent manner, whereas NDMAOAc did not modulate IL-12 production (Figure 3). Both end products, keto alcohol and keto acid, significantly (P < 0.05) inhibited AM IL-12 release (Figure 3). These results suggest that the pathway implicated in DNA pyridyloxobutylation and keto alcohol are responsible for IL-12 inhibition induced by NNK activation. The end product keto acid, formed from {alpha}-hydroxymethylene-NNK, is also implicated in the modulation of AM IL-12 production. However, the pathway implicated in the DNA methylation had no effect on the modulation of IL-12 production.



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Fig. 3. Modulation of AM IL-12 production. AM were treated for 20 h with NNK and different concentrations of NNKOAc or NDMAOAc in the presence of BCG (5 x 106 CFU/ml). NNK, NNKOAc, keto acid (KA) and keto alcohol (KAL) significantly (*P < 0.05, **P < 0.005) inhibited IL-12 production whereas NDMDOAc had no effect. Means ± SEM of five experiments.

 
Similar experiments were performed to identify the activation pathway responsible for the modulatory effect of NNK on AM NO production. AM were treated as mentioned above in the presence of LPS (10 ng/ml) for 48 h. NNKOAc significantly (P < 0.001) inhibited AM NO release in a concentration-dependent manner, whereas NDMDOAc had no effect. The end products, keto alcohol and keto acid, also had no effect on NO production (Figure 4). These results suggest that the pyridyloxobutylation intermediate is involved in the inhibition of NO production by NNK.



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Fig. 4. Modulation of AM NO production. AM were treated for 48 h with NNK and different concentrations of NNKOAc or NDMAOAc in the presence of LPS (10 ng/ml). NNK and NNKOAc significantly (*P < 0.05 and {dagger}P < 0.001, respectively) inhibited NO production whereas NDMAOAc, keto acid (KA) and keto alcohol (KAL) had no effect. Means ± SEM of eight experiments.

 
Modulation of anti-inflammatory mediators
To determine which activation pathway was involved in NNK-stimulation of AM IL-10 production, AM were treated as mentioned above, with LPS for 20 h. NNKOAc significantly inhibited (P < 0.001) in a concentration-dependent manner the production of IL-10 by AM, whereas NDMAOAc had no effect (Figure 5). Furthermore, keto acid and keto alcohol had no effect on the IL-10 production by AM.



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Fig. 5. Modulation of AM IL-10 production. AM were treated for 20 h with NNK and different concentrations of NNKOAc or NDMAOAc in the presence of LPS (10 ng/ml). NNK significantly (*P<0.05) stimulated IL-10 production, whereas NNKOAc significantly ({dagger}P < 0.001) inhibited it. NDMAOAc, keto acid (KA) and keto alcohol (KAL) had no effect on AM IL-10 release. Mean ± SEM of 10 experiments.

 
To further investigate the mechanism involved in NNK stimulation of IL-10 production, AM were treated with NNAL, a carcinogenic metabolite formed by the carbonyl reduction of NNK (Figure 1). Given that approximately half of the initial NNK is transformed into NNAL in NR8383 (28), AM were treated with 500 µM NNAL in the same conditions as mentioned above. NNAL had no effect on AM IL-10 production (data not shown). AM were also treated with NNKOAc, NDMAOAc and NNAL, alone and in combination, to investigate if the stimulation of IL-10 by NNK was caused by the synergy of several metabolites. All treatments containing NNKOAc significantly (P < 0.005) inhibited AM IL-10 production, whereas the NDMAOAc and NNAL combination had no effect (Table I). None of the precursors nor the end products used could explain the stimulation of AM IL-10 release by NNK.


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Table I. Modulation of alveolar macrophage (AM) IL-10 production

 
To further characterize the modulation of anti-inflammatory mediators by NNK, we investigated the effect of NNK, NNKOAc, NDMAOAc, NNAL, keto acid (KA) and keto alcohol (KAL) on PGE2 release. AM were treated with LPS (10 ng/ml) and NNK (500 µM), NNKOAc (50 µM) NDMAOAc (50 µM), KA (15 µM), KAL (15 µM) and NNAL (500 µM), for 3 h and supernatants were assessed for PGE2 production. NNK and NNKOAc significantly stimulated PGE2 production (P < 0.005 and P < 0.01, respectively) (Figure 6). NDMAOAc, KA and KAL had no effect on AM PGE2 production (data not shown).



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Fig. 6. Modulation of AM PGE2 production. AM were treated for 3 h with NNK, NNKOAc and NDMAOAc in the presence of LPS (10 ng/ml). NNK and NNKOAc significantly ({dagger}P < 0.001 and **P < 0.01, respectively) stimulated PGE2 production. NDMAOAc, keto acid (KA) and keto alcohol (KAL) had no effect on AM PGE2 release. Mean ± SEM of eight experiments.

 
To determine the role of PGE2 in the modulation of IL-10 release by NNK and NNKOAc, we analyzed the production of IL-10 when AM were treated with exogenous PGE2. AM were treated with a combination of NNK (500 µM) and PGE2 (10 µM) with LPS for 20 h and supernatants were assessed for IL-10 production. NNK, PGE2 and the combination of both significantly increased IL-10 production by 1.9-, 2.9- and 4.6-fold compared with sham-treated AM. These results suggest that the stimulation of IL-10 may occur via the PGE2 pathway. Given that PGE2 production is dependent on the COX pathway, COX-1 and COX-2, we investigated the effect of NNK and its metabolites on both COX-1 and -2 production. AM were treated with NNK for 1 h without LPS and COX-1 and -2 production was analyzed by flow cytometry. There was no significant modulation of COX-1 production when cells were treated with NNK and NNKOAc (data not shown). However, the production of COX-2 was significantly increased when cells were treated with NNK (500 µM) and NNKOAc (50 µM) (Figure 7). Given that IL-10 was measured after 20 h treatment with NNK, the COX-2 level was also investigated at that time. Under these conditions, NNK increased the expression of COX-2, whereas NNKOAc inhibited it (data not shown).



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Fig. 7. Modulation of AM COX-2 production. AM were treated for 1 h with NNK (500 µM) and NNKOAc (50 µM) without LPS. NNK and NNKOAc increased COX-2 expression (*P < 0.05). Mean ± SEM of four experiments.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
We have demonstrated previously that NNK, in addition to its carcinogenic effect, suppresses the production of pro-inflammatory mediators by AM (28). In the present study, we have determined the relative contribution of the two {alpha}-hydroxylation pathways to the modulation of AM mediator production. The first pathway investigated was the {alpha}-methylhydroxylation pathway leading to DNA pyridyloxobutylation and formation of keto alcohol, a non-carcinogenic metabolite. The results showed that this pathway is involved in the inhibition of inflammatory cytokine and mediator production caused by NNK exposure. This pathway is well known to be involved in DNA adducts formation (33). Recent studies have also shown that pyridyloxobutylated DNA in human lung is higher in cancer patients than in control subjects and that this pathway is critical in carcinogenesis by NNK (10,33,34). Thus, in addition to its carcinogenic activity, the {alpha}-methylhydroxylation pathway may contribute to the high incidence of lung cancer and respiratory infections in smokers or people exposed to second-hand smoke by modulating AM functions.

The NDMAOAc had no effect on AM mediator production, suggesting that NNK activation through the {alpha}-methylenehydroxylation pathway does not modulate the immune response. In contrast, this pathway is implicated highly in NNK carcinogenesis. Thus, the carcinogenicity mediated by the {alpha}-methylenehydroxylation pathway may not involve the modulation of AM cytokine production. However, modulation of other AM functions by this pathway remains to be investigated.

Keto alcohol and keto acid are non-carcinogenic end products of NNK metabolism. Keto acid, formed from a further oxidation of the keto aldehyde yielded from {alpha}-hydroxymethylene-NNK, has no carcinogenic effect (10). However, keto acid inhibited IL-12 production by AM, thus indicating that it may modulate AM function even though it has no carcinogenic activity. Keto alcohol, also a non-carcinogenic metabolite, is formed from the activation pathway leading to DNA pyridyloxobutylation by the reaction of diazohydroxide with H2O. Keto alcohol may be involved in the modulatory effect of NNK by inhibiting AM TNF and IL-12 production. Inhibition of these cytokines could contribute to the higher incidence of cancers and respiratory infections in smokers (20,35), IL-12 and TNF being implicated in tumoricidal activity and inflammation. Given that keto alcohol is a final product of the pathway leading to DNA pyridyloxobutylation, the inhibitory effect on pro-inflammatory cytokine production caused by the {alpha}-methylhydroxylation pathway may be mediated, in part, by the end product of the pathway instead of the reactive intermediate. However, the inhibition of NO production could not be explained by the effect of keto alcohol. Given the importance of the {alpha}-methylhydroxylation of NNK in the formation of DNA adducts and its role in the immunomodulation of AM, it will be of great interest to find a way to inhibit this pathway or at least to decrease its activation.

Our results indicate that NNK metabolism through the {alpha}-methylhydroxylation pathway is implicated in the inhibition of pro-inflammatory mediators such as TNF, IL-12 and NO. An inhibition of these pro-inflammatory mediators may reduce the defence mechanism of AM against tumoral cells and bacterial invasion (20,3638). IL-12 inhibition leads to a decrease in interferon-{gamma} (IFN-{gamma}) production, thus reducing T-cell proliferation and differentiation towards Th1 and cytotoxicity mediated by natural killer cells (18,19,39). An inhibition of IL-12 could decrease AM functions and activities in immune response, thus reducing AM capacity to protect lung homeostasis.

TNF and NO inhibition by NNKOAc and NNK may also be involved in the increase of lung cancer and respiratory infection in smokers. TNF participates to host defence by increasing cell phagocytosis, AM cytotoxic activity (40,41), and the production of other cytokines such as IL-1, IL-6 and IFN-{gamma} (42), whereas NO is one of the cytotoxic mediators produced by AM, helping the clearance of tumor cells and bacteria in the lung (43). All these events are necessary to protect the organism against foreign invasion (44,45). These results indicate that, even though NNK carcinogenicity is mediated by DNA methylation and pyridyloxobutylation caused by the reactive metabolites formed by its activation, the end products such as keto alcohol, could also participate in NNK carcinogenicity by modifying lung homeostasis and immune response. Thus, the carcinogenicity of NNK is caused, at least in part, by the modulation of cytokine production, contributing to tumor cells spreading.

The stimulation of AM IL-10 production by NNK could not be explained by any of the precursors or final products investigated. NNK being transformed in majority (~56%) in NNAL via the carbonyl reduction pathway (14), we tested the effect of NNAL and combination of precursors resulting from NNK activation to understand the stimulation of IL-10 by NNK. NNAL had no significant effect on IL-10 production by AM whether it was alone or in combination with NNKOAc, NDMAOAc or both. None of the precursors or final products used could explain the stimulation of AM IL-10 production by NNK, suggesting that this effect may be mediated by a different pathway and may involve aldehydes generated along the reactive alkylating intermediates.

NNK, NNKOAc and KAL stimulated AM PGE2 production. Exogenous PGE2 stimulated IL-10 production as demonstrated previously (27). Thus, the increase in IL-10 release might be mediated by the stimulation of PGE2 production by NNK. We also demonstrated that the stimulation of AM PGE2 production by NNK was mediated by COX-2. NNK stimulated COX-2 expression leading to an up-regulation of PGE2 production, which in turn may up-regulate IL-10 release by AM. Although NNKOAc up-regulated PGE2 production and COX-2 expression after 3 and 1 h treatment, respectively (Figures 6 and 7), it inhibited COX-2 expression and IL-10 after 20 h treatment. NNKOAc stimulation of PGE2 may not last long enough to up-regulate IL-10 production by AM as measured 20 h later. To better understand the inhibitory effect of NNKOAc on AM IL-10 production, cells were stimulated with exogenous PGE2 in the presence of NNKOAc and LPS for 20 h. Even in the presence of PGE2, which stimulates IL-10 production, NNKOAc significantly inhibited AM IL-10 production (data not shown). This result suggests that there is a different pathway implicated in the inhibitory effect of NNKOAc on AM IL-10 production.

Taken together, these results suggest that NNK alone, without any metabolic activation, could be responsible for the stimulation of IL-10. A previous study had already reported that NNK is a strong agonist competing successfully with nicotine to bind nicotinic acetylcholine receptors (nAChRs) (46,47). The affinity of NNK for {alpha}7 nAChR is ~1300 times greater than nicotine. Thus, NNK may stimulate IL-10 production via binding to nicotinic receptor without any metabolic activation. The presence of nAChRs was demonstrated in the central and peripheral nervous system and on many other tissue cells throughout the body including immune cells (48). Furthermore, nicotine has been demonstrated to modulate AM activities, such as anti-microbial activity and cytokine responses, through nAChRs (49). However, further investigations are needed to understand the mechanism involved in the stimulation of AM IL-10 production by NNK, which could be initiated by the binding of NNK to nAChRs.

In summary, NNK activation inhibits AM TNF, IL-12 and NO production but increases IL-10 and PGE2 release. These data suggest that NNK favors the differentiation of AM into polarized type 2 cells (AM-2) (50,51). Interestingly, this type of macrophage has been associated with tumor growth and progression (50). Furthermore, the reduction of type 1 AM, which is important for killing of tumor cells and microorganisms, may limit host defence capacity of the smokers and second-hand exposed subjects. This study indicates that the {alpha}-methylhydroxylation pathway of NNK, in addition to its DNA adduct formation via pyridyloxobutylation of DNA, is involved in the immunomodulation of AM cytokine production by NNK. The other hydroxylation pathway, the {alpha}-methylenehydroxy pathway, leading to the formation of methylated DNA, is likely to have a lesser important role in this immunomodulation. However, further studies are needed to better understand the implication of the immunomodulatory effects of NNK and its metabolites on AM functions and to determine a way to decrease the metabolic activation of NNK to prevent DNA adduct formation and reduce its immunomodulatory effect on AM.


    Acknowledgments
 
The authors thank Véronique Turmel for her excellent technical support and Sylvie Pilote for the HPLC analysis. This study was supported by the Canadian Institutes of Health Research. E.Y.B. is a senior FRSQ scholar and L.I.P. has a studentship from Réseau en Santé Respiratoire du FRSQ.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 28, 2003; revised January 20, 2004; accepted January 26, 2004.