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

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, alpha-methylhydroxylation and alpha-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 E(2) (PGE(2)) production by A549 cells, its immunomodulatory effects were not mediated by PGE(2) 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.

PCT-233, a novel modulator of pro- and anti-inflammatory cytokine production.

Plant extracts have been implicated in various immunoregulatory effects that are poorly understood. Thus, we investigated the modulatory activity of PureCell Complex (PCT)-233, an active molecular complex from mesophyll tissue of Spinacia oleacea on the inflammatory process. Alveolar macrophages (AM) were treated with PCT-233 and/or budesonide, a well-known anti-inflammatory agent, before or after being stimulated with lipopolysaccharides (LPS). Pro- and anti-inflammatory cytokine production, tumour necrosis factor (TNF) and interleukin (IL)-10, respectively, were measured in cell-free supernatants at different times after the treatment. PCT-233 increased unstimulated AM release of both TNF and IL-10, whereas heat- and light-inactivated PCT-233 stimulated only the release of TNF without affecting IL-10 production, suggesting that different mechanisms are involved in the modulation of TNF and IL-10 release by PCT-233. The presence of LPS did not modify PCT-233-stimulated TNF production, but the ratio TNF/IL-10 production by LPS-stimulated AM was reduced significantly in the presence of PCT-233. Pretreatment of AM with PCT-233 and budesonide before LPS stimulation reduced TNF production at both protein and mRNA levels, whereas IL-10 production was increased. Moreover, TNF/IL-10 ratio was reduced further with the combination PCT-233/budesonide. Interestingly, AM treatment with PCT-233 and budesonide 18 h after LPS stimulation did not modulate TNF release significantly but it did increase IL-10 production, and a synergistic effect was observed with the combination PCT-233/budesonide. These exciting data suggest that PCT-233 possesses some anti-inflammatory properties, even when added during the inflammatory process, and could potentiate the effect of other anti-inflammatory agents.

Immunomodulatory effects of the tobacco-specific carcinogen, NNK, on alveolar macrophages.

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.