Objective: Expression of tumor necrosis factor alpha (TNF), TNF receptors 1 and 2 and TNFalpha converting enzyme (TACE) was studied in A549 human alveolar epithelial cells exposed to organic dust from a swine barn. Additional objectives were to elucidate whether 8-bromocAMP affected TNF and TNF receptor mRNA expression by activation of protein kinase A (PKA) and whether it increased phosphorylation of cAMP-responsive element-binding protein (CREB).
Materials and methods: Reverse transcriptase- (RT-) PCR was performed on unexposed cells and cells exposed to a dust-suspension, with and without 8-bromo-cAMP (1 mM). H-89 was used to inhibit PKA. To further investigate mRNA expression of TNF, staurosporine was used. Immunolabeling was applied for detection of TNF, TNFR1, TNFR2 and phosphorylation of CREB.
Results: TNF mRNA and protein was expressed after 1-3 h in dust-exposed cells. TNFR2 mRNA and protein expression was induced by dust-exposure, whereas expression of TNFR1 and TACE was constitutive. After 1-1.5 h incubation, mRNA expression of TNF was (PKA-independently) attenuated by 8-bromo-cAMP (p < 0.05), whereas that of TNFR1 was PKA-dependently stimulated (p < 0.05). Staurosporine attenuated mRNA expression of TNF (p < 0.05), but not interleukin (IL)-6, which was detected prior to TNF.
Conclusion: Expression of TNF and its receptors in alveolar epithelial cells may contribute to the response to organic dust. 8-bromo-cAMP, which increased the number of cells exhibiting phosphorylation of CREB exerted opposite effects on TNF and TNFR1 mRNA expression. The mechanism by which cAMP attenuates TNF mRNA expression remains to be established. Dust-induced expression of IL-6 precedes that of TNF and the induction pathways differ with regard to staurosporine sensitivity.