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, 132 (2), 232-8

Immunomodulatory Effects of the Tobacco-Specific Carcinogen, NNK, on Alveolar Macrophages

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Immunomodulatory Effects of the Tobacco-Specific Carcinogen, NNK, on Alveolar Macrophages

M-J Therriault et al. Clin Exp Immunol.

Abstract

Lung cancer is strongly associated with cigarette smoking. More than 20 lung carcinogens have been identified in cigarette smoke and one of the most abundant is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). We hypothesized that NNK modulates alveolar macrophage (AM) mediator production, thus contributing to carcinogenesis. An AM cell line, NR8383, was treated with [3H]NNK and lipopolysaccharide (LPS), and NNK metabolites released in supernatants were analysed by high-performance liquid chromatography (HPLC). NNK was metabolized by carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol (NNAL) or activated by alpha-carbon hydroxylation. AMs were also treated with NNK (100-1000 micro M), with and without LPS, for different periods of time (6-72 h), and mediators released in supernatants were quantified by enzyme-linked immunosorbent assay (ELISA) or the Griess reaction. NNK inhibited (in a concentration-dependent manner) AM production of tumour necrosis factor (TNF), macrophage inflammatory protein-1alpha (MIP-1alpha), interleukin (IL)-12 and nitric oxide (NO), whereas IL-10 production was increased. Cyclooxygenase inhibitors - NS-398 and indomethacin - and anti-prostaglandin E2 (anti-PGE2) antibody abrogated the NNK-inhibitory effect on MIP-1alpha production by AM. NNK stimulated the release of PGE2, and exogenous PGE2 inhibited AM MIP-1alpha production, suggesting that the NNK immunomodulatory effect may be mediated by PGE2 production. Thus, in addition to its carcinogenic effects, NNK may contribute to the lung immunosuppression observed in tobacco smokers.

Figures

Fig. 1
Fig. 1
Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by the rat alveolar macrophage cell line, NR8383. Structures in square brackets are hypothetical.
Fig. 2
Fig. 2
Inhibition of alveolar macrophage (AM) production of macrophage inflammatory protein-1α (MIP-1α) by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). AMs were treated for 20 h with NNK (100–1000 µM), without (a) or with (b) lipopolysaccharide (LPS) (10 ng/ml) and cell-free supernatants were tested for MIP-1α. Spontaneous release of MIP-1α was significantly inhibited (*P < 0·05, †P < 0·005) in a concentration-dependent manner. Furthermore, NNK (1000 µM) significantly (†P < 0·005) inhibited LPS-stimulated MIP-1α release. The mean values ± standard error of the mean (s.e.m.) are shown of seven to 11 experiments.
Fig. 3
Fig. 3
Time-course analysis of macrophage inflammatory protein-1α (MIP-1α) inhibition by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (a) and modulation of MIP-1α mRNA expression (b).(a) Alveolar macrophages (AMs) were treated with NNK (1000 µM), with or without lipopolysaccharide (LPS) (10 ng/ml), for increasing periods of time (6, 20 and 48 h). Maximum inhibition was observed at 48 h (29·2%) and 20 h (52·7%) when AMs were treated with or without LPS, respectively (*P < 0·05, †P < 0·005). Mean values ± standard error of the mean (s.e.m.) of four to 11 experiments are shown. (b) AMs were treated with 500 µM NNK for 2 h followed by a further 2 h of incubation with or without LPS (10 ng/ml), RNA was isolated and reverse transcription–polymerase chain reaction (RT–PCR) performed to assess mRNA expression of MIP-1α and β-actin (housekeeping gene). NNK inhibited mRNA expression in both LPS-stimulated and unstimulated AMs. One representative experiment out of three is shown.
Fig. 3
Fig. 3
Time-course analysis of macrophage inflammatory protein-1α (MIP-1α) inhibition by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (a) and modulation of MIP-1α mRNA expression (b).(a) Alveolar macrophages (AMs) were treated with NNK (1000 µM), with or without lipopolysaccharide (LPS) (10 ng/ml), for increasing periods of time (6, 20 and 48 h). Maximum inhibition was observed at 48 h (29·2%) and 20 h (52·7%) when AMs were treated with or without LPS, respectively (*P < 0·05, †P < 0·005). Mean values ± standard error of the mean (s.e.m.) of four to 11 experiments are shown. (b) AMs were treated with 500 µM NNK for 2 h followed by a further 2 h of incubation with or without LPS (10 ng/ml), RNA was isolated and reverse transcription–polymerase chain reaction (RT–PCR) performed to assess mRNA expression of MIP-1α and β-actin (housekeeping gene). NNK inhibited mRNA expression in both LPS-stimulated and unstimulated AMs. One representative experiment out of three is shown.
Fig. 4
Fig. 4
Inhibition of alveolar macrophage (AM) mediator production by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). AMs were treated for 20 h with NNK in the presence of lipopolysaccharide (LPS) (10 ng/ml) for the release of tumour necrosis factor (TNF) (a) or in the presence of bacille Calmette–Guérin (BCG) for the release of interleukin-12 (IL-12) b). NNK significantly inhibited (†P < 0·005, ‡P < 0·001 and *P < 0·05) the production of tumour necrosis factor (TNF) and IL-12 by AMs. Mean values ± standard error of the mean (s.e.m.) of five to eight experiments are shown. (c) AMs were treated with NNK for 48 h in the presence of LPS (10 ng/ml) for the release of nitric oxide (NO). NNK significantly inhibited (*P < 0·05 and ‡P < 0·001) AM NO production in a concentration-dependent manner. Mean values ± standard error of the mean (s.e.m.) of 13 experiments are shown.
Fig. 5
Fig. 5
Increase of alveolar macrophage (AM) interleukin-10 (IL-10) production by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). AMs were treated with NNK (100–1000 µM) in the presence of lipopolysaccharide (LPS) (10 ng/ml) for 20 h, and IL-10 was measured in cell-free supernatants. NNK significantly (*P < 0·05) increased IL-10 release by AMs. Mean values ± standard error of the mean (s.e.m.) of six experiments are shown.
Fig. 6
Fig. 6
Mediator implicated in the inhibition of alveolar macrophage (AM) macrophage inflammatory protein-1α (MIP-1α) production by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). AMs were treated with NNK (500 µM), alone or together with NS-398 (10 µM), indomethacin (10 µM) or anti-prostaglandin E2 (anti-PGE2) antibody (dilution 1 : 25), or with PGE2 (25 ng/ml) without lipopolysaccharide (LPS), for 20 h. Cell-free supernatants were analysed for MIP-1α content. COX inhibitors and anti-PGE2 antibody abrogated the inhibitory effect of NNK on MIP-1α release by AMs (no significant inhibition). NNK and exogenous PGE2 significantly (*P < 0·005) inhibited AM MIP-1α release.
Fig. 7
Fig. 7
Modulation of alveolar macrophage (AM) prostaglandin E2 (PGE2) production by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). AMs were treated with NNK (500 µM) for 2 h in the presence or absence of lipopolysaccharide (LPS) (1 ng/ml), and PGE2 levels were measured in cell-free supernatants. NNK significantly increased (†P < 0·005 and ‡P < 0·0005) PGE2 release by unstimulated and LPS-stimulated AMs. Mean values ± standard error of the mean (s.e.m.) of eight experiments are shown.

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