Although cigarette smoke extract (CSE) is widely used in respiratory research, the methods used in its preparation are diverse and there is no consensus as to whether cigarette smoke extract (CSE) induces a pro-inflammatory or an immunosuppressive response in epithelial cells.1 Although the adverse effect that cigarette smoke has on the lower airway has been extensively studied, the responses of the nasal epithelium are not well described. Our aims were to study the effects of a non-cytotoxic CSE exposure on LPS-induced innate immune responses from primary nasal epithelial cells (PNECs), to assess the constitutive gene expression and the localisation of TLR-4 in PNECs by RT-PCR and flow cytometry, and finally to analyse the modulation of TLR-4 expression after stimulation PNECs by LPS±CSE pre-treatment. CSE was prepared by combusting 1 or 2 12 mg tar Marlboro cigarettes through 25 ml of media. Submerged PNEC cultures were treated with CSE (or vehicle) followed by stimulation with LPS. Supernatants were assessed for IL-8 and IL-6 and the expression and localisation of TLR-4 was established. Cell viability was not affected except after 24 h incubation with 5% CSE made from two cigarettes, or using single cigarette along with a 24 h stimulation with 20–25 μg/ml LPS. A brief incubation with CSE (4 h) significantly inhibited LPS-induced cytokine release. In contrast, a more prolonged incubation with CSE (24 h) heightened LPS-induced cytokine release. Incubation with CSE (4 h and 24 h) had no significant impact on expression of TLR4 mRNA. A brief incubation with CSE (4 h) resulted in a lower percentage of surface and intracellular TLR4. A prolonged incubation with CSE (24 h) did not affect surface or intracellular TLR4. CSE exposure for 4 h suppresses the inflammatory response in PNECs to stimulation with LPS whereas 24 h CSE exposure was pro-inflammatory. A reduced surface TLR-4 may account for the immunosuppressive effects, but unaltered surface and intracellular TLR-4 after 24 h CSE treatment suggests that alternative pathways may be responsible for the pro-inflammatory effects.
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