Cigarette smoking induces profound suppression of T cell responses in the lungs, but peripheral responses are not affected. We have identified four compounds in cigarette smoke that, at doses found in a single cigarette, block discrete steps in T lymphocyte activation and cell cycle progression. Acrolein and crotonaldehyde are volatile alpha, beta-unsaturated carbonyl compounds in the gas phase of cigarette smoke; they inhibit production of several proinflammatory cytokines, including IL-2, TNF-alpha and IFN-gamma, which are critical for T cell responses. Acrolein and crotonaldehyde suppress cytokine gene expression by inhibiting induction of NF-kappaB and AP-1 DNA binding activities. Hydroquinone (HQ) and catechol are present in the particulate fraction. They do not have significant effects on cytokine production and allow partial progression from G0 to G1 following T cell activation. However, they inhibit normal entry and progression through the G1 phase of the cell cycle. The localized nature of smoking-induced immune suppression is due to the fact that only the lungs and adjacent lymph nodes are subjected to immunosuppressive levels of these compounds. The long-term objective of this project is to identify the molecular mechanisms by which cigarette smoke suppresses T cell responses. Towards this goal, we propose the following specific aims: (1) to determine the effects of acrolein and crotonaldehyde on induction of NF-kappaB and AP-1 complexes and their ability to interact with their respective target promoters; (2) to determine the mechanism by which HQ and catechol regulate E2F-mediated gene expression and thereby control entry and progression through the G1 phase of the cell cycle; and (3) to assess the effects of HQ and catechol on c-myc transcription initiation and elongation. Human peripheral blood or lymph node lymphocytes (from non-smoking cadaveric organ donors) will be isolated and treated with cigarette smoke extracts, HQ, catechol, acrolein or crotonaldehyde, then stimulated with anti-CD3 + anti-CD28 or PMA. Whole cell, cytoplasmic or nuclear extracts of these cells will be analyzed for NF-kappaB and AP-1 by Western blot and mobility shift assays. Analysis of cell cycle proteins (Myc, Cdk4, Cdk6, cyclins, E2F4, p130) will be performed by flow cytometry, immunoprecipitation and western blot techniques. Myc RNA will be measured by real time PCR and nuclear run-on assays.
