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. 2005 Apr;140(1):46–53. doi: 10.1111/j.1365-2249.2005.02739.x

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, a component of tobacco smoke, modulates mediator release from human bronchial and alveolar epithelial cells

L I Proulx *, M Gaudreault *, V Turmel *, L A Augusto *, A Castonguay †, É Y Bissonnette *
PMCID: PMC1809332  PMID: 15762874

Abstract

Respiratory epithelial cells are known to contribute to immune responses through the release of mediators. The aim of this study was to characterize the immunomodulatory effects of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco carcinogen, on respiratory epithelial cells and to compare two metabolic pathways, α-methylhydroxylation and α-methylenehydroxylation, involved in these effects using selective precursors, 4-(acetoxy-methylnitrosamino)-1-(3-pyridil)-1-butanone (NNKOAc) and N-nitroso (acetoxymethyl) methylamine (NDMAOAc), respectively. Human bronchial and alveolar epithelial cell lines, BEAS-2B and A549, respectively, were treated with NNK, NNKOAc and NDMAOAc for 24 h with and without tumour necrosis factor (TNF) and mediators released in cell-free supernatants were measured by enzyme-linked immunosorbent assay (ELISA). NNK significantly inhibited interleukin (IL)-8, IL-6 and monocyte chemoattractant protein-1 (MCP-1) production in both cell types. Similar results were observed with primary bronchial and alveolar epithelial cells. Although NNK increased prostaglandin E2 (PGE2) production by A549 cells, its immunomodulatory effects were not mediated by PGE2 according to the results with cyclo-oxygenase inhibitors. NNKOAc mimicked NNK effects, whereas NDMAOAc significantly inhibited IL-8 production in BEAS-2B cells and MCP-1 in both cell types. These results demonstrate that NNK and its reactive metabolites have immunosuppressive effects on respiratory epithelial cells, which could contribute to the increased respiratory infections observed in smokers and the development and/or the progression of lung cancer.

Keywords: cytokines, chemokines, α-hydroxylation, human lung epithelial cells

Introduction

In the early 1990s, cigarette smoking was declared the leading cause of preventable death in United States by the Centers for Disease Control [1]. Cigarette smoking is associated with an increased incidence of airway infection and numerous diseases including lung cancer and chronic obstructive pulmonary disease (COPD) [2–4].

Tobacco smoke contains more than 4000 compounds including at least 20 substances known to induce lung cancer in laboratory animals [3]. Among those, the nicotine-derived 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most abundant nitrosamine in the human environment and the most potent and specific carcinogen for lung tissues [5]. NNK requires cellular metabolism to perform its tumorigenic activities [3,6]. NNK is activated by three different pathways namely, carbonyl reduction, pyridine N-oxydation and α-hydroxylation [6]. Two α-hydroxylation pathways are possible: α-methylhydroxylation and α-methylenehydroxylation generating simultaneously electrophilic carcinogenic intermediates. Both pathways are catalysed by specific cytochrome P450, lipoxygenases and cyclo-oxygenases [7,8]. The α-methylhydroxylation pathway leads to the formation of a keto alcohol and a reactive intermediate that pyridyloxobutylates DNA, whereas the α-methylenehydroxylation pathway yields keto acid and a reactive intermediate that methylates DNA [6]. The role of each pathway in NNK immunomodulatory effects can be characterized using two precursors, 4-[(acetoxymethyl)-nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) and N-nitroso(acetoxymethyl)methylamine (NDMAOAc), yielding exclusively the two reactive intermediates generated by α-methylhydroxylation and α-methylenehydroxylation pathways, respectively.

Tobacco smoke impairs the immune responses and alters many cell types in the lung, including pulmonary epithelial cells [9–11]. These cells have long been known for their role as a protective barrier against the external environment, but there is growing evidence suggesting that they also modulate the immune response [12]. Interestingly, cigarette smoke has been shown to increase the production of interleukin (IL)-1β, IL-6, IL-8, and decrease the secretion of transforming growth factor (TGF)-β and fibronectin from bronchial epithelial cells [11,13–16]. Epithelial permeability is increased by cigarette smoke extract, whereas bronchial epithelial cell proliferation, chemotaxis and their ability to remodel extracellular matrix are inhibited [11,17]. Although several studies have demonstrated the effects of cigarette smoke on pulmonary epithelial cells, the cigarette smoke component responsible for these effects is still unknown. Thus, we hypothesized that NNK, one component of tobacco smoke, contributes to the alterations of bronchial and alveolar epithelial cell functions contributing to the altered immune response observed in smokers.

BEAS-2B cells, a human bronchial epithelial cell line, and A549 cells, a human alveolar epithelial cell line, have been used extensively in previous studies to investigate the effect of cigarette smoke on inflammatory response and toxicity [15,16,18]. Thus, these cell lines were used to characterize the effects of NNK on the production of pro- and anti-inflammatory mediators. Given that these are transformed cell lines, primary normal lung epithelial cell lines were used to confirm the data.

Materials and methods

Chemicals

NNK (99% pure) and NDMAOAc (99·8% pure) were purchased from Chemsyn Science Laboratory (Lenexa, KS, USA) and LKT laboratories (St Paul, MN, USA), respectively. NNKOAc and NNK metabolites (keto acid and keto alcohol) were synthesized as described previously [19].

Cell culture

The human bronchial epithelial cell line, BEAS-2B, has been immortalized by transformation with the 12-SV40 virus hybrid and was purchased from ATCC (Manassas, VA, USA). The BEAS-2B cells were kept in culture in LHC-9 medium (Biosource Int., Camarillo, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Medicorp Inc., Montreal, QC, Canada), 1% Hepes buffer (Invitrogen, Burlington, ON, Canada) and 1% penicillin–streptomycin (Invitrogen), 0·2% gentamicyn (Sabex, Quebec, QC, Canada). A549 cells (gift from Dr J. Couet, Laval University) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented as described above. Normal human bronchial epithelial cells (NBHE) and small airway epithelial cells (SAEC) were purchased from Cambrex (Walkersville, MD, USA) and maintained in culture as suggested by the supplier. All cell lines were kept at 37°C in a humidified 5% CO2 atmosphere. The cells were trypsinized using 0·12% trypsin (Invitrogen).

Epithelial cells were treated with NNK (500 µm), NNKOAc (15 µm), NDMAOAc (15 µm), keto alcohol (15 µm) and keto acid (15 µm) for 24 h and cell-free supernatants were recovered and stored at −70°C for future analysis. The concentrations used were based on NNK metabolism by alveolar macrophages and cell viability [20,21]. In some cases, cells were simultaneously treated with NNK and human tumour necrosis factor (TNF; 25 ng/ml) (Peprotech Inc., Rocky Hill, NJ, USA). Cell viability was determined using Trypan blue dye exclusion method.

Mediator production

Levels of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) were measured in cell-free supernatants using enzyme-linked immunosorbent assay (ELISA) (BD Bioscience, San Jose, CA, USA) with a sensitivity of 4 pg/ml, 2 pg/ml and 4 pg/ml, respectively. The level of prostaglandin E2 (PGE2) was measured in cell free supernatants using an enzyme immunoassay (EIA) kit from Cayman Chemical Co (Ann Harbor, MI, USA) having a sensitivity of 7 pg/ml.

Reverse transcriptase-polymerase chain reaction (RT-PCR)

BEAS-2B and A549 cells were treated with NNK (500 µm) with and without TNF (25 ng/ml) for 24 h. Total RNA was extracted using TRIzol reagent (Invitrogen) and quantified using RiboGreen™ quantification reagent (Molecular Probes Inc, Eugene, OR, USA). Readings were performed on a Fluoroskan Ascent FL (Labsystems, Franklin, MA, USA). For cDNA synthesis, 1 µg of total RNA was reverse transcribed by Moloney murine leukaemia virus reverse transcriptase enzyme (Invitrogen) using a Minicycler™ (MJ Research, Waltham, MA, USA) according to the manufacturer's protocol. Polymerase chain reaction (PCR) was performed using Promega Taq DNA polymerase and the annealing temperature used for all primers was 55°C. The primers used were (1) human IL-6 sense 5′-ATG TAG CCG CCC CAC ACA GA-3′ and antisense 5′-CAT CCA TCT TTT TCA GCC AT-3′ (187 bp); (2) human IL-8 sense 5′-ATT TCT GCA GCT CTG TGT GAA-3′ and antisense 5′-TGA ATT CTC AGC CCT CTT CAA-3′ (255 bp); and (3) human GAPDH sense 5′-ATG CAA CGG ATT TGG TCG TAT-3′ and antisense 5′-TCT CGC TCC TGG AAG ATG GTG-3′ (221 bp). PCR products were run on a 2% agarose gel and stained with ethidium bromide (5 µg/ml).

Statistical analysis

Analysis of variance (anova) combined with Student's t-test for paired data were used to compare treatments. Differences were considered significant when P < 0·05.

Results

Inhibition of IL-8 secretion

To investigate the modulatory effects of NNK and the activation pathways implicated, A549 and BEAS-2B cells were treated for 24 h with NNK (500 µm), NNKOAc (15 µm) and NDMAOAc (15 µm) in the presence or absence of TNF (25 ng/ml). These concentrations did not affect cell viability as assessed by Trypan blue exclusion (data not shown). NNK significantly inhibited the IL-8 secretion of unstimulated and TNF-stimulated BEAS-2B cells (Fig. 1a). NNKOAc and NDMAOAc showed similar inhibition, suggesting that both types of α-hydroxylation are involved in the modulation of IL-8 by NNK. The end products, keto acid and keto alcohol, also inhibited the secretion of IL-8 in BEAS-2B cells (data not shown), suggesting that they may participate to the modulation of the immune response.

Fig. 1.

Fig. 1

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Inhibition of lung epithelial cell IL-8 production by NNK and related compounds. Cells were treated for 24 h with NNK (500 µm), NNKOAc (15 µm) or NDMAOAc (15 µm) with and without TNF (hatched and filled bar, respectively) and IL-8 assayed in cell-free supernatants. Left axis is for IL-8 values of unstimulated cells and the right one for TNF-stimulated cells. BEAS-2B TNF-stimulated IL-8 release was inhibited significantly by all compounds (‡P < 0·005, **P < 0·01 and *P < 0·05) compared with control (a), whereas A549 IL-8 release was significantly (‡P < 0·005) inhibited by NNK and NNKOAc (b). Means ± s.e.m. of six experiments.

A549 cells released spontaneously more IL-8 than BEAS-2B cells (1624 ± 93 pg/106 cells and 290 ± 17 pg/106 cells, respectively). Both spontaneous and TNF-stimulated IL-8 releases were significantly inhibited by the presence of NNK (Fig. 1b). NNKOAc significantly inhibited A549 IL-8 release, but NDMAOAc did not modulate IL-8 release, suggesting the implication of the α-methylhydroxylation pathway in this modulation. The end product of this pathway, keto alcohol, also significantly inhibited the secretion of IL-8 by A549 cells (data not shown).

To explore further the mechanism by which NNK modulates IL-8 production, we investigated the effect of NNK on IL-8 mRNA level. BEAS-2B and A549 cells were treated with NNK for 24 h in the presence or absence of TNF and RNA was isolated to perform RT-PCR. NNK treatment reduced the expression of IL-8 mRNA in both cell types, although the inhibition was stronger in BEAS-2B cells compared with A549 cells (Fig. 2a). NNK significantly inhibited TNF-stimulated IL-8 mRNA expression in both cell lines as demonstrated by the densitometry analysis (Fig. 2b).

Fig. 2.

Fig. 2

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Inhibition of IL-8 mRNA expression in sham and NNK (500 µm)-treated BEAS-2B and A549 cells. RNA was isolated after 24 h treatment and RT-PCR performed to assess mRNA expression of IL-8 as well as GAPDH (housekeeping gene). Results of a representative experiment are presented in (a). Relative mRNA expression of IL-8 was quantified by densitometry analysis normalized against GAPDH and mean ± s.e.m. of five experiments are presented in (b). *P < 0·05.

Inhibition of IL-6 secretion

To characterize the modulation of NNK on IL-6 secretion, BEAS-2B cells were treated as mentioned above. NNK significantly inhibited IL-6 release from both unstimulated and TNF-stimulated BEAS-2B cells (Fig. 3a). Given the low IL-6 release from unstimulated BEAS-2B cells, TNF-stimulated cells were treated with NNKOAc and NDMAOAc to investigate the pathway involved in this inhibition. NNKOAc significantly reduced IL-6 secretion of TNF-stimulated BEAS-2B cells. However, NDMAOAc and the two end products, keto acid and keto alcohol, did not significantly modulate TNF-stimulated IL-6 secretion (data not shown). These data suggest that the reactive intermediate of the α-methylhydroxylation pathway may be involved in the modulation of IL-6 production by bronchial epithelial cells.

Fig. 3.

Fig. 3

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Inhibition of lung epithelial cell IL-6 production by NNK and related compounds. Cells were treated for 24 h with NNK (500 µm), NNKOAc (15 µm) or NDMAOAc (15 µm), with and without TNF (hatched and filled bar, respectively), and IL-6 assayed in cell-free supernatants. Left axis is for IL-6-values of unstimulated cells and the right one for TNF-stimulated cells. IL-6 spontaneous release was significantly reduced by NNK (‡P < 0·005) and BEAS-2B TNF-stimulated IL-6 release was significantly inhibited by NNK (†<0·001) and NNKOAc (‡P < 0·005) (a). A549 spontaneous release of IL-6 was significantly inhibited by NNK (†P < 0·001) (b). TNF-stimulated release of IL-6 was significantly inhibited by both NNK and NNKOAc (‡P < 0·005). Means ± s.e.m. of six experiments.

Unstimulated A549 cells produced low but measurable amounts of IL-6, which was inhibited by NNK (Fig. 3b). TNF-stimulated A549 produced significantly more IL-6 than unstimulated cells (734·9 ± 51·8 pg/106 cells and 21·6 ± 1·6 pg/106 cells, respectively). Thus, A549 were stimulated with TNF (25 ng/ml) in the presence of NNK, NNKOAc, NDMAOAc, keto alcohol and keto acid. NNK and NNKOAc significantly inhibited the secretion of IL-6 in TNF-stimulated A549 cells, whereas NDMAOAc (Fig. 3b) and the end products (data not shown) did not significantly modulate IL-6 release from these cells.

To further explore the modulation of IL-6 production by NNK, IL-6 mRNA level was investigated. BEAS-2B and A549 cells were treated with NNK for 24 h in the presence or absence of TNF, and RNA was isolated to perform RT-PCR (Fig. 4a). NNK reduced the expression of IL-6 mRNA by 27% and 73% in BEAS-2B and A549 cells, respectively, when they were stimulated with TNF (Fig. 4b). Unstimulated lung epithelial cells expressed too low a level of IL-6 mRNA to be measured by densitometry analysis.

Fig. 4.

Fig. 4

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Inhibition of IL-6 mRNA expression in sham and NNK (500 µm) treated BEAS-2B and A549 cells. RNA was isolated after 24 h treatment and RT-PCR performed to assess mRNA expression of IL-6 as well as GAPDH (housekeeping gene). Results of a representative experiment are presented in (a). Relative mRNA expression of IL-6 was quantified by densitometry analysis normalized against GAPDH and mean ± s.e.m. of five experiments are presented in (b). *P < 0·05.

NNK modulation of mediator production

Bronchial epithelial cells, BEAS-2B, spontaneously released measurable amount of MCP-1 (161·0 ± 31·9 pg/106 cells), but this release was not modulated significantly by NNK treatment (132·6 ± 30·1 pg/106 cells). TNF stimulation increased MCP-1 release and NNK reduced this production significantly by 14% (2688 ± 532 pg/106 cells and 2282 ± 401 pg/106 cells, respectively). Alveolar epithelial cells, A549, produced significantly more MCP-1 (15607 ± 1053 pg/ml) than BEAS-2B cells and NNK treatment significantly inhibited spontaneous and TNF-stimulated MCP-1 production (Fig. 5). Moreover, both NNKOAc and NDMAOAc significantly inhibited TNF-stimulated MCP-1 release, suggesting that metabolites of NNK α-hydroxylation are important in the inhibition of MCP-1 production.

Fig. 5.

Fig. 5

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Inhibition of MCP-1 production by NNK. A549 cells were treated for 24 h with NNK (500 µm), with and without TNF (hatched and filled bar, respectively) and MCP-1 was assayed in cell-free supernatants. Left axis is for MCP-1-values of unstimulated cells and the right one for TNF-stimulated cells. NNK significantly inhibited both spontaneous and TNF-stimulated MCP-1 release (†P < 0·001). Both NNKOAc and NDMAOAc significantly inhibited TNF-stimulated MCP-1 production (†P < 0·001). Means ± s.e.m. of six experiments.

Other inflammatory mediators such as nitric oxide (NO), TNF and granulocyte-macrophage colony stimulating factor (GM-CSF) were also investigated. However, NO and TNF levels were below the assay sensitivity, whereas the release of GM-CSF was not significantly modulated by NNK (data not shown).

The production of anti-inflammatory cytokines such as TGF-β and IL-10 was also investigated. However, NNK did not significantly modulate the production of TGF-β by A549 and BEAS-2B cells, whereas IL-10 production was too low to be measured (data not shown).

NNK chronic exposure

To evaluate the cumulative effect of NNK exposure, cells were treated for 5 days with low concentration of NNK (7 µm), corresponding approximately to the amount found in one pack of cigarettes, and cytokine levels were measured. Overnight treatment of cells with low concentration of NNK (100 µm) did not significantly modulate cytokine release by airway epithelial cells (data not shown). However, when 7 µm NNK was added every day for 5 days (given a total concentration of 35 µm NNK), significant inhibition of IL-6, IL-8 and MCP-1 release by both A549 and BEAS-2B cells was observed (Table 1). Thus, exposure to repeated low concentration of NNK (7 µm for 5 days) gave similar results than a short-term treatment (24 h) with high NNK concentration (500 µm).

Table 1.

Modulation of cytokine production by low NNK concentration for 5 days.

A549 BEAS-2B
Treatment IL-6 IL-8 MCP-1 IL-6 IL-8 MCP-1
Sham 7·5 ± 0·2 888·7 ± 60·8 10347·8 ± 482·7 13·8 ± 1·9 47·8 ± 1·8 262·8 ± 9·5
NNK 4·4 ± 0·1† 534·7 ± 24·9†  4530·1 ± 199·7†  9·0 ± 1·0† 30·8 ± 0·8† 179·2 ± 8·3†
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Cells were treated with NNK (7 µm) every day for 5 days and cytokine levels were measured in cell-free supernatants (pg/106 cells). NNK significantly (†P < 0·001) inhibited the release of IL-6, IL-8, and MCP-1 by both A549 and BEAS-2B cells.

PGE2 production

We have demonstrated previously that the immunomodulatory effects of NNK on alveolar macrophages were mediated by PGE2 production [20,21]. Thus, PGE2 production by airway epithelial cells treated with NNK was investigated. TNF did not increase PGE2 release, but NNK significantly stimulated the release of PGE2 by TNF-stimulated A549 cells (Fig. 6). An increase of PGE2 release was also observed in unstimulated cells, but it did not reach significance. In contrast, PGE2 production by BEAS-2B cells was not modulated by NNK treatment (data not shown).

Fig. 6.

Fig. 6

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Increase of PGE2 production by NNK. A549 cells were treated with NNK (500 µm) for 3 h in the presence or absence of TNF and the PGE2 level was measured in cell-free supernatants. NNK significantly stimulated (*P < 0·05) the release of PGE2. Means ± s.e.m. of six experiments. Hatched bars, presence of TNF; black bars, absence of TNF.

To investigate the role of PGE2 in NNK modulation of cytokine production, A549 and BEAS-2B cells were treated with NNK in the presence of cyclo-oxygenase (COX) inhibitors (indomethacin and NS-398, 2 and 1 µm, respectively) and cytokine production was measured in cell-free supernatants. NNK inhibition of IL-6, IL-8 and MCP-1 was not abrogated by COX inhibitors (data not shown), suggesting that PGE2 is not involved in this inhibition.

Normal airway epithelial cells

Given that BEAS-2B and A-549 are transformed cell lines, the modulation of IL-8 and IL-6 production by NNK was investigated in normal human bronchial epithelial cells (NHBE) and small airway epithelial cells (SAEC) to confirm our data. NHBE cells released less IL-8 and IL-6 than SAEC when stimulated with TNF (Fig. 7). NNK significantly inhibited IL-8 and IL-6 release from both NHBE and SAEC cells, confirming our data obtained with transformed cell lines.

Fig. 7.

Fig. 7

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Inhibition of IL-8 and IL-6 production in lung primary epithelial cells, NHBE and SAEC, by NNK. Cells were treated for 24 h with NNK (500 µm) plus TNF, and IL-8 and IL-6 were assayed in cell-free supernatants. NNK significantly inhibited IL-8 and IL-6 production (*P < 0·05) in both cell types. Means ± s.e.m. of five experiments.

Discussion

NNK is one of the most abundant carcinogens found in cigarette smoke [3]. Although its role in lung cancer development and/or progression is well documented [3,5–7], little is known about its effects on immune responses. Recently, we have demonstrated that NNK inhibits the production of TNF, macrophage inflammatory protein-1α (MIP-1α), IL-12 and NO from alveolar macrophages, but increases the release of IL-10 and PGE2 [20,21]. Our data suggest that, in addition to its carcinogenic activity, NNK may contribute to lung immunosuppression observed in tobacco smokers.

In the past decade, pulmonary epithelial cells have emerged as central modulators of the inflammatory response [12]. Thus, to understand better the role of NNK on the immune response, we investigated its effects on the release of mediators by transformed human bronchial and alveolar epithelial cells, BEAS-2B and A549, respectively. We demonstrated that NNK inhibits IL-8 production from these cells at both protein and mRNA levels (Figs 1 and 2). Similar results were observed with primary normal lung epithelial cells, NHBE and SAEC. IL-8 is a chemokine that plays an important role in acute inflammation by recruiting and activating neutrophils [22] and in lung repair [23]. A reduction of IL-8 production may contribute to the enhanced incidence in lung infection observed in smokers [2]. In contrast, several studies have demonstrated an increase of IL-8 production in pulmonary epithelial cells exposed to cigarette smoke extract [14–16]. However, a study by Rusznak et al. demonstrated that short exposure (20 min) to cigarette smoke increases IL-8 release, whereas longer exposure (1–6 h) reduced it [24], as we observed. A recent study has also demonstrated an inhibition of IL-8 production by primary human alveolar type II epithelial cells when exposed to cigarette smoke extract [25]. These differences may be due, at least in part, to the variability in the composition of cigarette smoke extracts and NNK concentration in it. Indeed, NNK levels vary in different cigarette brands [26], which could also explain the controversy of IL-8 level in bronchoalveolar lavage fluids of smokers [27,28]. Furthermore, IL-8 in bronchoalveolar lavage may origin from different cell types such as alveolar macrophages. NNK may differently modulate IL-8 production depending on cell types. Thus, the reduction of IL-8 production by NNK, one component of cigarette smoke, may reduce the inflammatory response and repair of injury caused by cigarette smoking.

The modulation of IL-6 production by cigarette smoke is still unclear. In vitro studies with pulmonary epithelial cells showed a small increase in IL-6 release or no modulation [16], whereas an inhibition of IL-6 production was demonstrated in alveolar macrophages [29]. Furthermore, lower IL-6 level was observed in bronchoalveolar lavage of smokers compared with nonsmokers [28]. A decrease of IL-6 production may be involved in the reduction of natural killer cell activity and localized inflammation in cigarette smokers [30]. In the present study, we demonstrated that NNK inhibits the release of IL-6 from transformed and normal pulmonary epithelial cell lines (Figs 3 and 7b). Our data show the controversy of the modulation of this cytokine by tobacco smoke components. Distinct cell types may respond differently to tobacco smoke. Moreover, cell-to-cell communication between alveolar macrophages and pulmonary epithelial cells may be important in the modulation of cytokine production [31]. Further studies are needed to understand the communication between these cell types when exposed to NNK. This is currently under investigation in our laboratory.

MCP-1 is a chemoattractant protein for monocytes and it activates natural killer cell activity [32]. We demonstrated that NNK significantly down-regulates MCP-1 production of alveolar epithelial cells (Fig. 5) and TNF-stimulated bronchial epithelial cells. Furthermore, treatment of BEAS-2B to low concentration of NNK during 5 days caused a significant inhibition of MCP-1 spontaneous release, showing the importance of chronic exposure. Cigarette smoke extract has been shown to increase MCP-1 release from A549 cells [15], but to inhibit its release from primary human alveolar type II epithelial cells [25]. According to our data, one of the cigarette smoke components responsible for MCP-1 inhibition is NNK. The presence of other components in cigarette smoke extract may explain the difference obtained in other studies [15].

Our data showed that NNK increases the release of PGE2 by A549 cells. Although PGE2 is often considered as a proinflammatory mediator, it reduces the immune inflammatory response in the lung [33]. PGE2 inhibits the production of TNF and IL-12, whereas it increases IL-10 production [34]. However, the inhibition of IL-6, IL-8, and MCP-1 production by pulmonary epithelial cells was not mediated by PGE2 release, as demonstrated with COX inhibitors. Nevertheless, PGE2 inhibits tumour-cell apoptosis and induces tumour-cell proliferation [35]. Thus, the increase of PGE2 production by NNK may play an important role in cancer development.

Cultured alveolar epithelial cells have been shown to metabolize NNK [36]. Using metabolic precursors, we investigated the pathways involved in the modulation of cytokine production. Our data suggest that the α-methylhydroxylation of NNK is the pathway involved in the inhibition of cytokine production as we have demonstrated for alveolar macrophages [21]. Interestingly, α-methylenehydroxylation pathway is also involved in the inhibition of IL-8 in BEAS-2B cells, suggesting that these cells are more sensitive to the metabolites produced by this pathway. Although α-hydroxylation of NNK is involved in the modulation of mediator production by airway epithelial cells, other mechanism may be implicated in these effects. Indeed, NNK can bind to nicotinic acetylcholine receptors in human bronchial epithelial cells activating the PI3K/Akt pathway [37]. Some immunomodulatory effects of NNK may be mediated via these receptors. Further investigations are needed to understand the role of these receptors and the signal transduction pathways involved in the immunomodulatory effects of NNK.

There is some controversy with the use of transformed cell lines which may be differently modulated than normal cells. However, we have demonstrated that NNK cytokine modulation was similar in transformed cell lines, BEAS-2B and A-549, and primary cells, NHBE and SAEC. Thus, both types of cells may be used in further studies with NNK.

Although NNK did not significantly modulate the production of GM-CSF and TGF-β, tendencies toward a reduction of proinflammatory cytokines and an increase in anti-inflammatory mediators was observed. Thus, the overall effect of NNK on both bronchial and alveolar epithelial cells is immunosuppressive which may contribute to increased inflammatory disease incidence observed in smokers. Given the importance of cell-to-cell communication, it would be interesting to investigate whether the effect of NNK on pulmonary epithelial cell production of mediators can be modulated by the co-culture with inflammatory cells such as alveolar macrophages and neutrophils.

Acknowledgments

The authors thank Émilie Gélinas for her excellent technical support. This study was supported by the Canadian Institutes of Health Research. E.Y.B is a senior Fond de Recherche en Santé du Québec (FRSQ) scholar and L.I.P. has a studentship from Réseau en Santé Respiratoire du FRSQ.

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