Organic dust-induced signaling in human pulmonary epithelial cells : Emphasis on effects of cAMP modulation

Sammanfattning: To study signaling transductions in innate immunity a model system was applied where A549 alveolar epithelial type II cells were used as target and organic dust collected in swine confinement buildings as stimulus. The dust represents a complex mixture of feed and faecal particles, dander from swine, bacteria (whole bacteria and cell wall components) and fungi. Inhalation of the dust causes intense airway inflammation, which is unrelated to allergy and characterized by an influx of neutrophilic granulocytes, and systemic effects in previously unexposed healthy subjects. Swine farmers have increased prevalence of chronic airway diseases e.g. chronic bronchitis. Specific aims were to study if organic dust i) influences ion exchange, in particular the N +/H + exchanger (NHE), ii) activates G a i-coupled receptors resulting in decreased intracellular cAMP levels, iii) affects expression of tumor necrosis factor-alpha (TNF) and its receptors R1 and R2. Cyclic AMP generally provides a suppressive signal in inflammation and an additional aim was to iiii) elucidate effects of increased cAMP on expression of the pro-inflammatory cytokines interleukin (IL)-6, IL-8, TNF and the TNF receptors, R1 and R2 using 8-bromo-cAMP and the PKA-inhibitor, H-89. Expression of Toll-like receptors (TLRs) -2 and -4, and the antimicrobial peptides (AMPs), HBD2 and LL-37 was also investigated. Effects on ion exchange depended on the proliferative state of the cells, as assessed by microphysiometry with a Cytosensor . Thus, NHE was activated by perfusion with dust in low-density A549 cells, but blocked during perfusion of high density-cells. Regardless of cell density, NHE was active during recovery, which may result in acidification of the extracellular milieu (I). Measurements of cAMP accumulation after 30 min stimulation with organic dust indicated no activation of G a i-coupled receptors (I). No time-kinetic effects were revealed by measurement of cytokine release with ELISA, since only cumulative effects were recorded (II), but RT-PCR showed that dust-stimulated mRNA expression of IL-6 and TNF peaked after 1.5h incubation; on prolonged incubation expression of the mRNAs was subjected to negative feedback regulation. Furthermore, dust-induced mRNA expression of IL-6 was detected before that of TNF and the induction pathways differed with regard to staurosporine sensitivity. Expression of basal and dust-stimulated IL-8 mRNA was sustained for at least 24h. TNFR1 was constitutively expressed, but expression of TNFR2 mRNA and protein was induced by organic dust (III, IV). Incubation in dust together with 8-bromo-cAMP increased the number of cells exhibiting phosphorylation of the transcription factor CREB at Ser133 and time-kinetically shifted the relative contribution of the cytokines. Thus, d uring the first two hours 8-bromo-cAMP increased basal and dust-induced mRNA expression of IL-6, but attenuated expression of TNF mRNA. IL-8 mRNA expression was decreased by 8-bromo-cAMP during 1.5h-6h incubation in dust. On prolonged exposure, 8-bromo-cAMP attenuated the mRNA expression of IL-6, but not of IL-8. TNFR1 mRNA expression was stimulated by 8-bromo-cAMP. With regard to IL-6, IL-8 and TNFR1 the effects of 8-bromo-cAMP appeared to be mediated by PKA; the attenuation of TNF expression was PKA-independent (III, IV). The tyrosine kinase (TK) inhibitor genistein potentiated forskolin-stimulated cAMP accumulation to the same extent as the phospho-diesterase inhibitor, IBMX; effects of increased cAMP may therefore be mistaken for effects on TK-signaling when genistein is used to study signaling transductions (V). No unstimulated expression of the TLRs or AMPs was found in A549 pulmonary epithelial cells, but dust-induced expression of TLR2 mRNA was demonstrated after 5h incubation and expression of LL-37, but not of HBD2 mRNA, was indicated after stimulation for 1.5h (thesis).